bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 355: Increasing Muscle Mass Improves Vascular Function In Obese Mice
Authors: Qiu Shuiqing , Mintz James , Salet Christina , Nernpermpisooth Nitirut , Fulton David , Stepp David
Published: July 2021

Exercise improves endothelial function in obese patients. Salutatory effects of exercise include increases in muscle size and quality, reduction in fat mass and alterations of the plasma milieu. The relationships between these changes and improvements in vascular function are poorly defined.

Hypothesis

Increasing muscle mass by deletion of muscle growth negative regulator myostatin, improves vascular function in mesenteric arteries from obese
db/db
mice. Myostatin deletion increased muscle mass in both lean (gastrocnemius 57.93%, gluteus maximus 60.95%, triceps 57.64%) and obese mice (gastrocnemius 79.64%, gluteus maximus 112.32%, triceps 103.61%). Myostatin deletion increased muscle fiber size in lean and obese mice (p< 0.05) but had no significant effects on adipocyte size in obese mice. Fasting glucose, HbA1c and glucose tolerance were improved in obese myostatin null mice. Obese mice demonstrated superoxide-mediated impairment of ACh-induced vasodilation compared to lean mice. Deletion of myostatin in obese mice improved ACh-induced vasodilation in mesenteric arteries without effects in lean mice. This improvement was blunted by L-NAME. PGI
2
and EDHF mediated vasodilation were preserved in obese mice, and unaffected by myostatin deletion. Treatment with sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopoterin) restored impairment of vasodilation in obese mice, and this improvement was blocked by L-NAME. Taken together, these data suggested that increasing muscle mass by deletion of myostatin improved NO not PGI
2
or EDHF mediated vasodilation in obese mice.
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 399: Increased Gene Transcription via Ire1a/cJun Enhances Plasma ALT/AST in MTP Inhibited and MCD Diet Fed Mice
Authors: Josekutty Joby , Iqbal Jahangir , Iwawaki Takao , Kohno Kenji , Hussain M. Mahmood
Published: July 2021

Microsomal triglyceride transfer protein (MTP) is a target to reduce plasma lipids because of its indispensable role in triglyceride-rich lipoprotein biosynthesis. MTP inhibition in western diet fed mice decreased plasma triglycerides/cholesterol while increasing plasma alanine/aspartate aminotransferases (ALT/AST) and hepatic triglycerides/free cholesterol. Free cholesterol accumulated in the endoplasmic reticulum (ER) and mitochondria resulting in ER and oxidative stresses. Mechanistic studies revealed that MTP inhibition increased transcription of the
GPT/GOT1
genes through up-regulation of the IRE1α/cJun pathway leading to increased synthesis and release of ALT1/AST1. We also observed that IRE1α/cJun pathway is involved in augmenting plasma ALT/AST in mice fed methionine-choline deficient diet. Thus, transcriptional up-regulation of
GPT/GOT1
genes is a major mechanism elevating plasma transaminases under different conditions.
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.
Abstract 42: A Talin Mutant that Disrupts Talin-integrin Binding in Platelets Decelerates aIIbß3 Activation Without Pathological Bleeding
Authors: Stefanini Lucia , Snider Adam , Piatt Raymond , Petrich Brian , Bergmeier Wolfgang
Published: July 2021

Tight regulation of integrin affinity is a critical component of hemostasis. The final step of integrin inside-out activation is talin binding to two distinct sites within the integrin β cytoplasmic tail, an NPLY sequence and a membrane-proximal region (MPR). Selective deletion of talin1 from platelets (Tln1fl/flPf4-Cre+) completely prevents integrin activation and thrombus formation, and results in profound defects in hemostasis. Interestingly, platelet-specific expression of a talin1 mutant (L325R) that lacks the ability to bind the MPR, but can still bind the NPLY sequence, phenocopies talin1-knockout platelets. In this study we sought to analyze the effects on thrombosis and hemostasis of a platelet-specific talin1 mutation (W359A) that dramatically impairs binding to the NPLY sequence and does not rescue the talin1 deficiency in transfected cells.

Homozygous knock-in of talin(W359A) is embryonic lethal in mice. Thus, Tln1W359A/wt mice were crossed with Tln1fl/flPf4-Cre+ mice to generate Tln1W359A/flPf4-Cre+ (TlnWA) and Tln1wt/flPf4-Cre+ (Tlnwt) control mice. Expression of talin(W359A) in platelets partially rescued talin1 deficiency. Compared to Tlnwt controls, agonist-induced αIIbβ3 activation was reduced by ~50% and spreading on fibrinogen was only moderately impaired in TlnWA platelets. However, kinetic studies demonstrated decelerated αIIbβ3 activation in TlnWA platelets, which resulted in delayed aggregation under static conditions and reduced thrombus size at low shear rates. Interestingly, adhesion of TlnWA platelets to collagen at high shear rates was not significantly better than that of talin-deficient cells and TlnWA mice were completely protected from FeCl3-induced carotid artery occlusion. Lastly, in contrast to platelet-specific talin-knockout or TlnLR mice, TlnWA mice showed no detectable gastrointestinal bleeding and only modestly increased tail-bleeding times compared to littermate controls.

In conclusion, we demonstrate that the talin(W359A) mutation decelerates, but does not abolish, talin-dependent integrin activation in platelets. Our studies further suggest that this delay in integrin activation is protective from pathological vessel occlusion while it does not affect hemostasis in mice.
Abstract 422: Atherosclerosis and Rage Expression by Histology and Imaging in Hyperlipidemic Pigs
Authors: Johnson Lynne , Tekabe Yared , Kollaros Maria , Eng George M , Bhatia Ketan , Groner Rachel B , Krueger Christian G
Published: July 2021

Purpose and methods

To characterize atherosclerotic lesions and RAGE expression in a large animal model and investigate targeting RAGE for in-vivo imaging. Nine hyperlipidemic pigs at 12 mo of age were studied. LDL averaged 513 ± 74 mg/dL and final wt was 63.3 ± 12.4 kg. Pigs were injected with 16.3 ± 3.5 mCi of
99m
Tc anti-RAGE F(ab’)
2
and after 8 hr (blood pool clearance) underwent SPECT/CT imaging of the neck and thorax and then euthanized, the aorta including innominate, proximal carotids, and proximal ileofemoral branches and coronaries were dissected. Vascular tissue was divided into 36 anatomical segments: coronaries: 4 each x 3 = 12, aortic root: 3, aortic arch: 4, thoracic aorta: 3 and abdominal aorta: 3, each carotid and femoral: 2. Sections from each segment were counted and stained for H&E and RAGE. Scans were analyzed for tracer uptake in organs and vascular structures and counts converted in %ID. All sections were classified according to AHA criteria, % stenosis, and % RAGE staining was quantified. One HL pig received non-immune control antibody and 2 farm pigs were injected for blood pool clearance and SPECT imaging.

Results

On pathological examination 75/324 segments or 23% showed class 2 lesion or greater. All class III or IV lesions were in the coronaries, carotids, and femoral arteries and % stenosis ranged from 16 to 65%. In vivo biodistribution showed intense uptake of the antibody in the lungs (15 %ID) indicating high constitutive expression of RAGE and precluded visualizing the thoracic aorta. Heart uptake was low (1.3 x 10
-4
%ID) providing visualization of focal coronary uptake and the carotids were well seen. Focal tracer uptake was visualized on SPECT scans in these 2 vascular territories for all vessels with class III or IV lesions and uptake ranged from 4 -22 %ID x 10
-4
. Correlations were R = 0.85, P = 0.002 for %ID vs. %ID/g and R = 0.83, P = 0.002 for %ID/g vs. % RAGE staining.

Conclusions

There is variability in extent and severity of atherosclerotic disease in HL pigs at one year. RAGE staining is found in all class II-IV lesions and correlated with uptake of radiolabeled antibody targeting RAGE. High RAGE expression in lungs limited imaging the aorta but carotid and coronary artery uptake was seen on SPECT scans.
Abstract 43: The Role of Epsins in Atherosclerosis
Authors: Chen Hong
Published: July 2021

Background

Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. We have recently published that epsins function as regulators of tumor angiogenesis by controlling the endocytosis of VEGFR2 (
J Clin Invest.
2012). The goal of this project is to define the novel role of epsins in endothelial cells (EC) in regulating inflammation-mediated atherogenesis.

Methods and results

To examine the role of endothelial epsins in atherogenesis, we engineered mice with constitutive deletion of epsins in EC (EC-DKO). Strikingly, these mice are viable but display markedly attenuated atherogenesis on an ApoE null background in response to hypercholesterolemia. Oil red O staining revealed that ApoE-/- mice with endothelial deletion of epsins had a significant reduction in atherosclerotic lesion area in aortic root and macrophage infiltration compared to ApoE-/- mice after being fed a western diet for 10 weeks. FACS analysis revealed that deletion of epsins greatly reduced TNFα and LPS-induced expression of adhesion molecules (ICAM & VCAM), selectins (P- & E-), CCR2 and MCP-1 in primary mouse aortic EC (MAEC) isolated from EC-DKO mice. Mechanistically, epsins promote TNFR/TLRs signaling and NF-κB and MAPK (JNK and p38) activation by stabilizing the receptor complex formation and enhancing recruitment of NEMO, a crucial player in NF-kB activation. Conversely, epsin deficiency does not affect the surface expression of TNFR1/TLR2/4 by FACS analysis, suggesting that epsins may be dispensable for endocytosis of TNFR1/TLR2/4. We also observed that a synthetic peptide comprising the epsin ubiquitin-interacting motif (UIM) potently disrupts the association of epsins with TNFR/TLR signaling complex in vitro, suggesting a potential pharmacological inhibitory role for this peptide in controlling the atherosclerosis in vivo. Our data demonstrate that epsins control atherogenesis by regulating TNFR/TLR signaling in endothelial cells and provide a potential novel strategy to perturb atherogenesis.

Conclusion

Our results demonstrated an essential role of epsins in atherogenesis by regulating TNFR/TLR signaling.
Abstract 435: Hepatic Carboxylesterase 1 is Essential for Maintaining Hepatic Triglyceride and Plasma Cholesterol Homeostasis and is Regulated by the Farnesoid X Receptor
Authors: Xu Jiesi , Li Yuanyuan , Xu Yang , Chen Wei-Dong , Yin Liya , Ge Xuemei , Jadhav Kavita , Adorini Luciano , Zhang Yanqiao
Published: July 2021

Non-alcoholic fatty liver disease (NAFLD) is one of the major health concerns worldwide, and is associated with increased risk of cardiovascular disease. Farnesoid X receptor (FXR) is considered a therapeutic target for treatment of NAFLD and lipid disorders. Here we investigated the role of hepatic carboxylesterase 1 (CES1) in lipid and carbohydrate metabolism. We found that the hepatic CES1 expression is affected by the status of glucose. Over-expression of hepatic CES1 lowered the levels of hepatic TG and plasma glucose in both wild-type and diabetic mice. Knockdown of hepatic CES1 increased both hepatic TG level and plasma cholesterol level. Such effects of CES1 likely resulted from its TG hydrolase activity, and subsequent changes in fatty acid oxidation and/or de novo lipogenesis. Hepatic CES1 was also inducible by FXR and was indispensable for activated FXR to improve both hepatic and plasma lipid homeostasis. Thus, hepatic CES1 plays a critical role in regulating lipid and carbohydrate metabolism and in FXR-controlled lipid homeostasis.
Abstract 44: Interleukin-27 Signaling is a Critical Regulator of Inflammation in Atherosclerosis
Authors: Koltsova Ekaterina , Kim Gisen , von Vietinghoff Sibylle , Kronenberg Mitchell , Ley Klaus
Published: July 2021

Atherosclerosis is chronic inflammatory disease, which affects blood vessels. While the pro-atherogenic role of various inflammatory cytokines was broadly investigated, less is known about contribution of anti-inflammatory cytokines with regard to their ability to control inflammation in vivo. Interleukin 27 (IL-27) was shown to play immunosuppressive function via multiple mechanisms. We tested whether IL-27 signaling is important to restrain inflammation in mouse models of atherosclerosis. We transplanted bone marrow from
Il27ra
-/-
or
Il27ra
+/+
mice into atherosclerosis prone
Ldlr
-/-
littermates. Recipients of
Il27ra
-/-
marrow showed significantly larger atherosclerotic lesions in aortic roots, aortic arches and, most strikingly, in the abdominal aorta. Aortas contained more CD45
+
leukocytes and CD4
+
T cells, which produced pro-atherogenic cytokines IL-17A and TNF-α. Concomitantly, the levels of IL-17A and IL-6 were significantly elevated in aortic tissue. These cytokines normally suppressed by IL-27, regulated the expression of CCL2 and other chemokines, which in turn led to accumulation of myeloid CD11b
+
and CD11c
+
cells in aortas, atherosclerotic plaque growth and disease progression. Moreover, using our recently developed live imaging by two-photon microscopy, we found enhanced interaction between antigen presenting cells and T cells in the arterial wall of
Il27ra
deficient mice. Overall, IL-27 signaling in bone marrow-derived cells regulates atherosclerosis by controlling interaction of antigen presenting cells and T cells in the arterial wall and therefore curbing Th17 and Th1 lineage differentiation, TNF and IL-17 dependent chemokine expression and subsequent myeloid cell accumulation. Thus, our work establishes the importance of anti-inflammatory cytokine signaling in atherosclerosis and demonstrates novel anti-atherogenic role of IL-27.
Abstract 452: Detailed Characterization of the Role of the Epidermal Growth Factor Precursor Homology Domain A of the Low-density Lipoprotein Receptor in Proprotein Convertase Subtilisin/kexin-type 9 Binding
Authors: Zhang Da-Wei , Adijiang Ayinuer , Gu Hong-mei
Published: July 2021

Proprotein convertase subtilisin/kexin-type 9 (PCSK9) promotes the LDL receptor (LDLR) degradation, thereby preventing clearance of LDL-C levels and playing a central regulatory role in cholesterol homeostasis. PCSK9 interacts with the LDLR at the cell surface and binds the receptor with a much higher affinity at the acidic environment of the endosome. Consequently, the receptor traffics from the endosome to the lysosome for degradation, rather than being recycled. Previously, we have shown that the epidermal growth factor precursor homology domain A (EGF-A) of the LDLR is critical for PCSK9 binding at the cell surface (pH7.4) and that leucine at the position 318 in the EGF-A is critical for PCSK9 binding. Here, we further characterized the role of EGF-A in the binding of PCSK9 to the LDLR in more details. We found that mutation of Asp299 to Val or Arg329 to Pro in the EGF-A significantly reduced PCSK9 binding at pH 7.4. In addition, we observed that deletion of EGF-A significantly reduced PCSK9 binding at the acidic endosomal environment (pH 6.0) and that PCSK9 bound to purified recombinant EGF-A in a pH-dependent manner with a stronger binding at pH6.0. However, mutation of Leu318 to Arg or His306 to Tyr that dramatically enhanced PCSK9 binding at pH7.4 had no significant effect on the binding of PCSK9 to the LDLR at pH6.0. Thus, our findings demonstrate that EGF-A of the LDLR plays an important role in PCSK9 binding at the cell surface (pH 7.4) and the endosomal environment (pH6.0). Our data also indicate that different amino acid residues in EGF-A are involved in PCSK9 binding to the LDLR at the cell surface and in the endosome.
Abstract 471: Triglyceride-rich Lipoprotein Lipolysis Products Induce Adipose Differentiation Related Protein Expression and Proatherogenic Cytokine Secretion in Monocytes
Authors: Norman Jennifer E , Aung Hnin H , Rutledge John C
Published: July 2021

Elevated blood triglyceride levels are associated with an increased risk of atherosclerotic cardiovascular disease. Blood triglycerides are contained within very low density lipoproteins (VLDL) and chylomicrons, which are referred to as triglyceride-rich lipoproteins (TGRL). The enzyme lipoprotein lipase is responsible for the hydrolysis of these triglycerides, primarily into free fatty acids and glycerol. Our lab has shown that these lipolysis products induce lipid droplet formation in monocytes and can induce a proinflammatory state in multiple cell types. Other investigators have linked adipose differentiation related protein (ADRP, also known as perilipin 2 or adipophilin) to lipid droplet formation in monocyte-derived macrophage foam cells. The objectives of this study are to further understand the effects of TGRL lipolysis products on the formation of lipid droplets in monocytes and monocyte activation. We hypothesize that TGRL lipolysis products increase the expression of ADRP and the secretion of proatherogenic cytokines in THP-1 monocytes. In this study, we analyzed the global gene expression in THP-1 monocytes exposed to TGRL lipolysis products. ADRP was identified as a principal response lipid droplet associated gene induced by exposure to TGRL lipolysis products when compared to the control treatment. Induction of protein expression was analyzed by western blot. The TGRL lipolysis product treatment was also found to induce genes associated with atherosclerosis and monocyte adherence to endothelium including growth differentiation factor 15 (GDF15) and interleukin 8 (IL-8) when compared to the control treatment. We measured the secretion of GDF15 and IL-8 by ELISA. Our results indicate that mRNA and protein expression of ADRP and secretion of proatherogenic cytokines (GDF15 and IL-8) were increased in monocytes after exposure to TGRL lipolysis products. In conclusion, our study demonstrates that TGRL lipolysis products induce the expression of ADRP and the secretion of proatherogenic cytokines in monocytes. These results may indicate a role for TGRL lipolysis products in the increased risk of atherosclerotic cardiovascular disease for individuals with elevated blood triglyceride levels.
Abstract 479: Expression of ApoE-sendai by Macrophages Causes Lipoprotein Glomerulopathy While Suppressing Atherosclerosis
Authors: Tavori Hagai , Yang Shilin , Fogo Agnes B , Fan Daping , Fazio Sergio
Published: July 2021

Renal disease is usually associated with lipoprotein abnormalities. Lipoprotein glomerulopathy (LPG) is a unique disease characterized by intraglomerular lipoprotein thrombi, often in the absence of dyslipidemia. Macrophages are not involved in lipid accumulation in LPG. ApoESendai is an apoE variant known to cause LPG in heterozygous carriers. It carries the same charge as the apoE2 common variant, but is due to a proline-for-arginine substitution at position 145 in the apoE3 sequence. The effects of macrophage expression of apoESendai on LPG and atherosclerosis have not been studied.We have characterized renal and arterial responses in apoE-/- or apoE-/-/LDLR-/- (DKO) mice expressing apoESendai exclusively in macrophages. Bone marrow from apoE-/- mice was transduced with lentiviral constructs expressing either human apoE2 or apoESendai, and then transplanted into apoE-/- or DKO recipient mice.Compared to BMT controls, both apoESendai and apoE2 macrophages significantly decreased plasma cholesterol levels in apoE-/- recipients (22.6% and 19.4%, respectively). No changes in serum cholesterol levels were found in DKO recipients compared to controls. Macrophage expression of apoESendai and apoE2 was associated with plasma human apoE levels of 6.9±1.9 and 5.4±1.7 μg/ml, respectively, in apoE-/- recipients, and 18.9±2.8 and 18.4±3.8 μg/ml, respectively, in DKO recipients. All mice displayed extensive kidney tubular lesions, which correlated with the severity of dyslipidemia. While none of the apoE-/- recipients showed any additional kidney damage, DKO mice expressing apoESendai showed clear features of LPG, with lesions in the glomerular capillaries characterized by mesangial cell expansion. Macrophage apoE2 expression by apoE-/- BMT recipients reduced aortic lesion size by 22.7% compare to BMT controls (p<0.01). Surprisingly, macrophage apoESendai expression further reduced aortic lesion size by 27.6% compared to apoE2 expression in apoE-/- mice (p<0.01). A similar 26.1% reduction was seen in DKO recipients transplanted with apoESendai compared to apoE2 expressing macrophages.Our results show for the first time that macrophage expression of apoESendai protects against atherosclerosis while causing glomerular disease.
Abstract 486: Platelet ITAM Signaling is Critical for Maintenance of Vascular Integrity in Inflammation
Authors: Boulaftali Yacine , Hess Paul R. , Getz Todd M. , Cholka Agnieszka , Stolla Moritz , Mackman Nigel , Owens A.Phillip , Ware Jerry , Kahn Mark L. , Bergmeier Wolfgang
Published: July 2021

Blood platelets, long recognized for their importance in primary hemostasis, have recently been identified as critical regulators of vascular integrity during inflammation and cancer. Interestingly, the contribution of platelets to the maintenance of vascular integrity in in[[Unable to Display Character: ﬂ]]ammation seems to be independent of their ability to form a clot. However, the underlying molecular mechanism remains unknown. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling but not G protein-coupled receptor (GPCR) signaling is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a novel protocol for the adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from wild-type platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of CLEC-2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into thrombocytopenic mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel anti-thrombotic agents directed towards the ITAM signaling pathway.
Abstract 5: A Novel APOE p.Leu166del Mutation in Autosomal Dominant Familial Hypercholesterolemia
Authors: Awan Zuhier , Choi Hong , Stitziel Nathan , Ruel Isabelle , Husa Regina , Gagnon Marie-Helene , Wang Rui Hao , Katherisan Sekar , Seidah Nabil , Genest Jacques
Published: July 2021

Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the low density lipoprotein receptor (LDLR), its ligand apoB (APOB) or in proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Yet DNA sequencing does not identify mutations in these genes in a significant number of cases with a phenotype of ADH, suggesting that ADH has multiple genetic etiologies. Through a combination of clinical examination, biochemical analysis, candidate gene sequencing, and next-generation exome sequencing, we identified an in-frame three base-pair deletion in apolipoprotein E (APOE, c.496_498delCTC) resulting in a novel Leu166del mutation. The proband presented with an acute myocardial infarction at age 43, requiring urgent coronary revascularization. He has extensive tendinous xanthomas and xanthelasmas, elevated levels of total cholesterol (11.2 mmol/L, 457 mg/dL)), LDL-C (9.69 mmol/L, 374 mg/dL), normal HDL-C (1.62 mmol/L, 63 mg/dL) and triglycerides levels (1.13 mmol/L, 100 mg/dL). A HPLC lipoprotein profile showed selective increase in LDL-C. One sister is heterozygous for the APOE Leu166del mutation and has an LDL-C of 5.3 mmol/L (180 mg/L). Another sister, with an LDL-C of 3.8 mmol/L (120 mg/dL) was not affected. DNA sequencing was performed for the LDLR, PCSK9, LDL-R adapter protein-1 (LDLRAP1) and exon 26 of the APOB genes no causal mutations were identified. We then performed exome sequencing on three individuals from the family. Approximately 54,000 variants were identified. We removed variants unlikely to be causal in the LDL-C genome-wide association studies, variants with a minor allele frequency >5%, synonymous or intronic variants and variants that did not fit a dominant model of transmission. We identified 49 missense mutations, 1 frameshift and 2 in-frame deletions in 52 genes. Using data derived from exome chip genotypes for association with LDL-C, the strongest evidence of association was found for the APOE gene. The Leu166del mutation is predicted to alter the protein structure of Apo E near the α-helix within the receptor binding domain. This report confirms that ADH can be caused by mutations within the APOE gene and represents the 4th loci causing ADH.
Abstract 500: Basal Peripheral Arterial Blood Flow Increases Concomitantly with Decreased Cardiopulmonary Reserve and Biomarkers Activation in Patients with Heart Failure and Preserved Ejection Fraction
Authors: Vitiello Damien , Harel François , Touyz Rhian M , Sirois Martin G , Lavoie Joel , Myers Jonathan , Gayda Mathieu , Chabot-Blanchet Malorie , Rouleau Jean-Lucien , De Denus Simon , White Michel
Published: July 2021

Background

The underlying pathophysiology of heart failure with preserved ejection fraction (HFpEF) remains poorly understood. Changes in peripheral arterial function concomitantly with the evaluation of cardiopulmonary reserve and biomarkers related to wall stress, extra-cellular matrix turnover (ECM), subclinical inflammation and oxidative stress have not been investigated in patients with HFpEF nor compared with age-matched healthy volunteers (HV). We hypothesized that patients with HFpEF would have impairments in cardiac reserve with reduced peripheral arterial function associated with broad-spectrum biomarkers activation.

Methods

Eighteen male and female HFpEF patients (aged 70 ± 9 yr) NYHA class II and III were recruited. Data from patients were compared with those from 14 age and sex matched HV. A maximal exercise testing with gas exchange analysis was completed on a treadmill using a RAMP protocol and heart rate recovery (HRR) was measured at 1 and 2 minutes following exercise. Peripheral arterial function was assessed using near infrared radionuclide plethysmography. Biomarkers included BNP, NT-proBNP, hsCRP, TBARS, 8-epi-prostaglandin F2α, MMP 1, 2, 9 and TIMP 1, 2, 3, 4) were analyzed.

Results

Selected data are presented in the Table 1 and 2.

Conclusions

Compared to healthy volunteers, patients with HFpEF demonstrated a significant decrease in aerobic capacity but an increase in basal peripheral arterial blood flow, subclinical inflammation and oxidative stress. The increase in resting arterial blood flow may be a compensatory mechanism for the decrease in cardiac reserve and the pro-inflammatory/oxidant milieu in these patients.
Abstract 508: Prolonged Thrombus Resolution Leading to Abnormal Collagen Fibrillogenesis and Angiogenesis in Injured Arteries of Type III Collagen-Deficient Mice: a Paradoxical Mechanism for ‘Tissue Fragility’ in Vascular Ehlers-Danlos Syndrome and Spontaneous Cervical Artery Dissection
Authors: Reid Amy J , Bai Yuqiang , Perez Lorenzo F , Ogawa Naomi , Buja L. M , Yeh Alvin T , Milewicz Dianna M
Published: July 2021

Patients with
COL3A1
mutations that impede secretion of type III collagen into matrix develop wounds that heal poorly following cutaneous injury. They are also predisposed to vascular complications like catastrophic rupture or dissection of cervical arteries and stroke. We sought to determine if low production of type III collagen would also interrupt arterial thrombus resolution, a process resembling wound healing, and whether defects contribute to risk of dissection. We injured cervical elastic arteries in mice by ligation of the left common carotid, halting proximal blood flow. Strikingly, injured arteries from

Col3a1
+/-

mice develop thrombi over three weeks that resist resolution more often than those in wild-type littermates (
p
=.002). The unresolved thrombi in

Col3a1
+/-

arteries also retain a higher burden of macrophages (
p
=.043) and proliferative α-actin-positive cells (
p
=.034), while mutant arteries display vascular channels within the media (
p
=.015) that are partially lined with endothelial cells, consistent with residual neoangiogenesis. At two weeks, burdens of macrophages (
p
=.009), proliferative cells (
p
=.028), and vascular channels (
p
=.019) are also higher in

Col3a1
+/-

arteries, despite actively resolving thrombi of equal length between the two groups (
p
=.338), suggesting that neoangiogenesis results specifically from lower
Col3a1
dose. Treating injured mice with rapamycin halts thrombus resolution in the early inflammatory phase of both genotypes and normalizes the incidence of vascular channels in

Col3a1
+/-

arteries at three weeks (
p
=1.00), suggesting that vascular channels result secondary to thrombus resolution and not from structural weakness in type III collagen deficient arteries. Patient-derived
COL3A1
mutant myofibroblasts in 3D culture models of fibrin remodeling accumulate less extracellular type I collagen following TGFβ1 stimulation but persistently upregulate expression of
Acta2
, relative to control cells whose
Acta2
expression levels peak and subside as collagen accumulates by seven days. These data implicate dysregulated thrombus remodeling in response to injury that increases the risk for medial neoangiogenesis, which may also increase the risk for dissection in affected arteries after injury.
Abstract 517: Effect of Hepatocyte-specific Depletion of AT1a Receptors on Atherosclerosis and Aneurysms
Authors: Rateri Debra L , Balakrishnan Anju , Howatt Deborah A , Moorleghen Jessica J , Cassis Lisa A , Daugherty Alan
Published: July 2021

Background and Objective

Whole body deficiency of AT1a receptors (AT1aRs) ablates angiotensin II (AngII) induced vascular pathologies that include aortic aneurysms and atherosclerosis. We created AT1aR floxed mice to determine the role of specific cell types expressing this receptor on these vascular pathologies. Unexpectedly, these mice demonstrated a lack of effect of smooth muscle specific AT1aR deficiency on the development of atherosclerosis or aneurysms. Also, endothelial-specific AT1aR deficiency had no effect in atherosclerosis and minimal effect on aneurysms. The purpose of this study was to determined if AngII infusion was releasing substances from the liver that influence vascular pathology.

Methods and Results

Hepatocyte depletion of AT1aRs was accomplished using two methods of introducing Cre into AT1aR floxed mice. One approach was injection of adenovirus Cre (AdCre) and the other by breeding to mice expressing Cre under the control of the albumin promoter. To induce atherosclerosis, groups of wild type and hepatocyte-specific AT1aR depleted male (N=15-21/group) and female (N=21/group) mice were fed a saturated fat enriched diet for 12 weeks. Hepatocyte-specific depletion of AT1aRs was confirmed using detection of Cre and absence of AT1aRs by PCR and RT-PCR, respectively. Body weights, serum cholesterol concentrations, and systemic blood pressures were not different between wild type and hepatocyte-specific AT1aR depleted mice grouped by gender and genotype. Atherosclerotic burden was similar in all groups. To examine AngII-induced atherosclerosis, male wild type and hepatocyte-specific AT1aR depleted mice (N=11-16/group) were fed a fat enriched diet for 5 weeks, and AngII (1,000 mg/kg/min) was infused using osmotic mini-pumps for 28 days. AngII infusion increased systolic blood pressure, promoted atherosclerosis and induced aneurysms to a similar degree in both genotypes.

Conclusion

Deletion of hepatocyte-specific AT1aRs did not affect dietary or AngII-induced vascular pathologies.
Abstract 531: Role of Rna Splicing In Mediating Lineage-specific Expression of the Von Willebrand Factor Gene in the Endothelium
Authors: Yuan Lei , Janes Lauren , Beeler David , Spokes Katherine C , Smith Joshua , Li Dan , Jaminet Shou-Ching , Oettgen Peter , Aird William C
Published: July 2021

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we employed transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human beta-globin, mouse DSCR-1 or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells, but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a post-transcriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.