bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 421: Biomarkers of Atherosclerotic Plaque Instability in Carotid Artery Stenosis
Authors: Lee Daniel J , Hislop Sean J , Doyle Adam J , Kumar Neil G , Roztocil Elisa , Gillespie David L , Cullen John P
Published: July 2021

Objective:
Atherosclerotic plaque instability has been linked to cardiovascular events. We hypothesized that novel serum and intraplaque markers of plaque instability could be identified through analysis of serum and atherosclerotic plaques obtained from symptomatic and asymptomatic patients undergoing carotid endarterectomy (CEA).

Methods:
Under an IRB approved protocol, 6 symptomatic patients (5 transient ischemic attack, 1 amaurosis fugax) and their age- and gender-matched controls were analyzed for intraplaque markers of plaque instability. 8 symptomatic patients and their age- and gender-matched controls were analyzed for serum activity of proteolytic enzymes MMP-2 and MMP-9. Plaques were removed intraoperatively, placed in RNAlater and processed immediately after CEA. Blood samples were drawn prior to incision to determine serum activity of MMP-2 and MMP-9 using gelatin zymography. Intraplaque expression of MMP-2, MMP-9, proteolytic enzymes (uPA, tPA and PAI-1) and inflammatory markers (TF, TSP-1, and TNF-α) were evaluated by Western blot analysis.

Results:
The activity of proteolytic enzymes MMP-2 and MMP-9 detected in serum was significantly higher in symptomatic patients when compared to asymptomatic controls, with a 1.5- and 2.2-fold increase, respectively (P<0.05, n=8). The activity of MMP-2 and MMP-9 detected in plaque was also elevated in the symptomatic group, with a 9.04 and 15.3-fold increase, respectively. Intraplaque expression of uPA (9.6-fold increase) and PAI-1 (28% decrease) was found in symptomatic patients. The expression of inflammatory markers TF and TSP-1 was elevated in symptomatic patients (2.7- and 4.2-fold, respectively) (All P<0.05, n=6). There was not a statistically significant difference in levels of tPA and TNF-α (P=0.075 and 0.345, respectively).

Conclusion:
Levels of matrix metalloproteinases, proteolytic enzymes and inflammatory markers found in serum and plaque samples of symptomatic and asymptomatic patients may be different. Testing for biomarkers may help diagnose atherosclerotic plaque instability and identify patients at high risk for cerebrovascular events.
Abstract 445: Phospholipid Levels Associated with Specific High-Density Lipoprotein Subfractions Predict Cardiovascular Risk in Young Adults with Type 2 Diabetes
Authors: Gordon Scott M , Shah Amy S , Lu L J , Deng Jingyuan , Dolan Lawrence M , Urbina Elaine M , Davidson W S
Published: July 2021

Risk for atherosclerosis is greatly increased in people who have type 2 diabetes (T2D). Because of this, the emerging epidemic of adolescent T2D holds ominous implications for premature cardiovascular disease (CVD). High density lipoprotein cholesterol (HDL-C) represents the body’s natural defense against CVD but its levels are depressed in individuals with T2D. Recent studies indicate HDL exists as distinct subspecies raising the possibility that certain species may be more cardioprotective than others. However, little is known regarding the role of HDL subspecies in T2D, especially in the adolescent population. Thus we sought to evaluate HDL subspecies and determine whether certain subspecies are associated with protection against the development of early atherosclerosis as measured by carotid intima medial thickness (IMT). Healthy controls and youth with T2D were recruited. Whole plasma was analyzed by high-resolution gel filtration chromatography to resolve HDL sized particles and lipids in each fraction were quantitated by colorimetric assay. T-tests were used to evaluate group differences and linear regression models were constructed to determine independent predictors of carotid IMT. Youth with T2D had higher BMI, total cholesterol and lower HDL-C compared to healthy controls, p<0.05. The groups did not differ in LDL-C, triglycerides or BP. Phospholipid distributions of HDL subspecies were found to be shifted in participants with T2D compared to controls (p<0.05). There was a significant inverse correlation between carotid IMT and the phospholipid content of larger HDL subfractions 22-24 (p<0.05) in youth with T2D. Linear models demonstrate HDL fraction 22 was the only independent predictor of carotid IMT while HDL-C, LDL-C, total -C and triglycerides were not significant. These data suggest an altered HDL particle subclass distribution may better predict protection against early atherosclerosis. Thus analyzing the HDL subspecies may be a more powerful approach to assessing cardiovascular risk than the currently accepted standard of HDL-C.
Abstract 481: Disturbed Flow Arterial Intimal Hyperplasia Model in the Mouse Carotid Artery Induced by a Focal Stenosis
Authors: Mauro Christine R , Tao Ming , Nguyen Binh , Yu Peng , Ozaki C K
Published: July 2021

Objective:
Murine models offer a power tool for the molecular dissection of remodeling and intimal hyperplasia mechanisms, though technical challenges limit their utility. While simple, complete carotid ligation lacks direct clinical relevance, and branch outflow ligations require advanced microsurgical skills. We thus developed a simple and clinically relevant mouse model based on the hypothesis that locally disturbed flow caused by a focal high grade stenosis would yield arterial intimal hyperplasia thickening, and that the standardized diet induced obesity (DIO) model would accentuate this response.

Methods:
A focal stenosis in 8 week old C57BL/6J mice (normal chow (NC, 10 kcal% fat) or DIO chow (60 kcal% fat) throughout study) was created by placing 9-0 nylon suture around the distal common carotid artery and an external 35-gauge needle (outer diameter=0.14mm), and then removing the needle to restore blood flow (∼78% reduction of lumen diameter; n=20). Tissues were perfusion fixed for morphology 4 weeks later.

Results:
Both NC and DIO groups developed intimal hyperplasia proximal to the stenosis, with approximately three-times more in the DIO animals (Figure 1).
Conclusions:
In the mouse, a surgically created focal stenosis yields an intimal hyperplastic wall response. DIO accentuates this response. This model offers a tool for investigating mechanisms of hemodynamically driven mechanisms governing intimal hyperplasia formation.
Abstract 497: Cholesterol and Toll-like Receptor Signaling Are Important Regulators of Macrophage Interactions with Atherosclerotic Lipoprotein Aggregates
Authors: Chin Harvey F , Haka Abigail , Maxfield Frederick R
Published: July 2021

Macrophages encounter deposits of aggregated low-density lipoproteins (agLDL) in the subendothelial space of blood vessels during the first stages of atherosclerotic plaque formation. Notably, current models for the mechanism of macrophage internalization of cholesterol in early atherosclerotic plaques are incomplete due to the lack of attention paid to the unique cellular mechanisms that are required for macrophages to degrade aggregates of LDL in particular, which can comprise >90% of the LDL in atherosclerotic plaques. In fact, internalization of cholesterol from cholesteryl esters in agLDL involves the development of intriguing cellular processes in which extracellular acidic compartments, lysosomal synapses (LSs), are formed whereby agLDL is partially degraded prior to internalization. This process requires extensive cytoskeletal rearrangements and secretion of lysosomal enzymes responsible for hydrolysis of cholesteryl esters from the agLDL. Subsequent delivery of free cholesterol from agLDL to the macrophage plasma membrane is central for development of the LS. Nonetheless, the molecular mechanism underlying initiation and propagation of the LS are currently largely unknown. This research proposal aims to elucidate the molecular mechanisms of LS formation and the role that cholesterol plays in eliciting these morphological responses to agLDL. Fluorescence microscopy assays were used to identify activation of TLR4 and downstream signaling involving PI3K and Akt as important events leading to LS formation. Furthermore, morphological responses of macrophages to cholesterol overloading require overlapping signaling pathways, indicating the role of interplay of cholesterol and TLR4 signaling in development of this novel macrophage interaction with aggregated LDL found in plaques. Identification of specific molecular pathways involved in this process will not only contribute to the basic understanding of one of the primary cellular processes contributing to atherosclerosis, one of the primary causes of heart disease, but also provide tangible molecular targets for the ultimate development of therapies.
Abstract 506: Hepatic Insulin Signaling Regulates ApoAI Expression Through Type I Deiodinase
Authors: Liu Jing , Hernandez-Ono Antonio , Galton Valerie A , Ginsberg Henry N
Published: July 2021

Low HDL cholesterol is a common feature of insulin resistant states such as type 2 diabetes, but little is known about the regulation of HDL cholesterol and apoAI levels by insulin signaling. In prior studies, we observed that liver insulin receptor (Insr) knockout mice (LIRKO) had very low plasma HDL cholesterol and apoAI levels compared with their controls. HDL cholesterol levels were normalized when we restored insulin signaling by expression of constitutively active (CA) AKT1. Acute knock down of hepatic Insr by adenovirus-mediated expression of albumin-Cre in Insr
flox/flox
mice resulted in a marked decrease in the levels of
ApoAI
and
Dio1
mRNA in the liver.
Dio1
encodes the Type 1 deiodinase (D1), which can convert thyroxine to 3,5,3’-triiodothyronine. Adenovirus mediated overexpression of
Dio1
increased HDL cholesterol and apoAI levels in LIRKO mice. D1 knockout mice exhibited a significant reduction in hepatic
ApoAI
mRNA levels. In McArdle cells, short interfering (si) RNA-mediated knockdown of Insr reduced both
Dio1
and
ApoAI
mRNA levels. Knockdown of Insr by siRNA reduced luciferase activity of both
hDio1
and
hApoAI
promoter constructs in HepG2 cells. Furthermore, siRNA-mediated knockdown of
Dio1
expression also decreased
hApoAI
luciferase activity. These findings indicate that insulin signaling regulates the expression of both
Dio1
and
ApoAI,
and that
Dio1
regulates
ApoAI
expression. Reductions in
ApoAI
gene expression may play a role in the etiology of low HDL cholesterol levels commonly present in states of insulin resistance. Targeting D1 may be a novel way to increase apoAI and HDL cholesterol levels in people with insulin resistance or type 2 diabetes mellitus.
Abstract 544: Extracellular Heat Shock Protein 27 Inhibits Foam Cell Formation by Targeting Scavenger Receptor-A in the Vessel Wall
Authors: Raizman Joshua E , Chen Yong-Xiang , Hu Thomas , Seibert Tara , Salari Samira , Hibbert Benjamin , Simard Trevor , Cuerrier Charles , Zhao Xiaoling , Ma Xiaoli , Rayner Katey , Moore Kathryn , O’Brien Edward
Published: July 2021

Background:
We showed that injection of recombinant heat shock protein 27 (rHSP27) into ApoE
-/-
mice reduces atherosclerosis lesions and total serum cholesterol levels. Extracellular HSP27 also interacts with scavenger receptor (SR)-A and inhibits acLDL uptake in macrophages, but the role of SR-A in HSP27 atheroprotection remains unclear.

Purpose:
To elucidate the mechanism of HSP27 atheroprotection by exploring if the presence of SR-A is required to facilitate its salutary effects.

Methods/Results:
rHSP27 treatment of bone-marrow derived macrophages from ApoE
-/-
mice reduced acLDL uptake (38%, p<0.05) using flow cytometry - effects not observed (p=0.171) in cells from ApoE
-/-
SR-A
-/-
mice. rHSP27 reduced acLDL binding (38%, p<0.01) and uptake (34%, p<0.01) in THP-1 macrophages associated with downregulation of SR-A transcript (46%, p<0.01) and total protein levels (63%, p<0.001). Flow cytometry and confocal microscopy revealed reduction in SR-A expression at the cell surface. To explore the signaling involved, NF-kB activation was assessed in THP-1 cells stably transfected with an NF-kB reporter. rHSP27 increased NF-kB activity (7 fold, p<0.001) and induced translocation of the NF-kB p65 subunit from the cytosol to the nucleus - effects that were inhibited (p<0.05) by the NF-kB antagonist, BAY. Addition of BAY inhibited the effect of rHSP27 on SR-A gene and protein expression and acLDL uptake (p<0.05). To explore the importance of SR-A in HSP27 atheroprotection, rHSP25 (mouse homologue) or PBS was administered to ApoE
-/-
and ApoE
-/-
SR-A
-/-
mice for 3 weeks on a high fat diet. Treatment of ApoE
-/-
mice revealed reductions of en face aortic lesions (39%, p<0.001), SR-A expression in the intima, and total serum cholesterol levels (32%, p<0.01). In the absence of SR-A, HSP27 atheroprotection was abolished (p=0.79) despite similar decreases in serum cholesterol levels (25%, p<0.05).

Conclusions:
Extracellular HSP27 inhibits foam cells by downregulating SR-A expression via an NF-κB-dependent mechanism. HSP27 atheroprotection requires the presence of SR-A expression in vivo. In the absence of SR-A, loss of atheroprotection occurs despite reductions in total serum cholesterol levels, suggesting SR-A as a novel target for HSP27 in the vessel wall.
Abstract 546: Physiologic Variations Between Hemoglobin Levels in Capillary and Venous Blood
Authors: Murphy William G , Browne Susan , Segurado Ricardo , Gallagher Brigid , Tong Emma , Murphy Ciaran , Grant Timothy
Published: July 2021

A complex relationship exists between venous hemoglobin level and thrombosis risk. The hemoglobin content of capillary blood varies in healthy individuals in relation to that of venous blood with sex, age, season of the year, and venous hemoglobin level. (Murphy WG, et al. Blood 2010;116:2861-2). To study further the dynamics of this relationship, venous and capillary (finger pulp blood) hemoglobin levels were compared in 462 frequent blood donors with hereditary hemochromatosis. A linear mixed effects model was fitted to compare capillary hemoglobin with the difference between the venous and capillary hemoglobin. Random slopes and intercepts for each donor were modelled to account for inter-individual variability. A linear mixed effects model was fitted to hemoglobin levels over cumulative time since first donation for each person in separate analyses for capillary and venous hemoglobin levels. Random intercepts and random slopes were modelled within individuals. For each change of 1g/dL in capillary hemoglobin, the difference between venous and capillary hemoglobin changed by -0.694 g/dL, significantly different from zero (p<0.001), indicating a dynamic relationship between the two compartments.

After venesection capillary hemoglobin rose by a mean of 0.496 mg/dL/day, (p<0.001) and venous hemoglobin by 0.113 mg/dL/day (p=0.019). The recovery rate for capillary hemoglobin between venesections was 4.39 times faster than for venous hemoglobin [t(4798)=2.743; p=0.006].

These data indicate that healthy individuals preserve venous hemoglobin at the expense of capillary hemoglobin as the red cell mass rises and falls, and that the capillary space buffers the venous hemoglobin level from changes in red cell mass. Since this space occupies > 10% of the intravascular volume (Chaplin H, et al. JCI 1953:32;1309-16) we estimate that 27% or more of a decrease or increase in red cell mass is accommodated by changes in hemoglobin level of capillary blood relative to the venous hemoglobin level.

The mechanisms underlying these dynamics, such as NO-induced vasodilation by deoxyhemoglobin and modulation of the Fåhraeus effect, remain to be elucidated; however their implications for the relationship between hemoglobin level and thrombosis may be considerable.
Abstract 10: Randomized, Double-Blind, Placebo-Controlled Single Ascending Dose Pharmacokinetic and Pharmacodynamic Study of PRT064445, a Universal Antidote for Factor Xa Inhibitors
Authors: Crowther Mark , Kitt Michael , McClure Matthew , Sinha Uma , Lu Genmin , Karbarz Mark , Hutchaleelaha Athiwat , Barron Lee , Mathur Vandana , Curnutte John
Published: July 2021

Background

Direct factor Xa inhibitors [fXaI] have superior anticoagulant efficacy and/or safety relative to warfarin and LMWH but are limited by lack of a specific antidote to reverse anticoagulation during episodes of serious bleeding or before surgery. PRT064445 [PRT] is a modified, human recombinant fXa that is catalytically inactive but retains high-affinity binding to direct fXaI and heparin- antithrombin III complexes. It competes with native fXa for fXaI drugs, thus reversing direct and indirect fXaI-mediated anticoagulation.

Methods

In this phase I, first-in-man double-blind study, 32 healthy volunteers were randomized (6:2) within dosing cohorts to a single IV bolus of PRT (30 mg, 90 mg, 300 mg, or 600 mg) or placebo and followed for 28 days. Anti-fXa activity was assayed in vitro by adding exogenous rivaroxaban (50 ng/mL) to subject plasma samples.

Results

The terminal t1/2 was ~6 hours. AUC and Cmax increased disproportionately relative to dose. In the presence of PRT, thrombin generation and anti-fXa activity of rivaroxaban (
Figure
) were reversed in a dose-dependent manner. There were no thrombotic AEs or deaths. There was 1 serious AE (pneumonia) and 3 non-serious infusion-related reactions without anaphylaxis [90 mg (2) and placebo (1)]. One unplanned pregnancy occurred ~10 days post-treatment, followed shortly by a spontaneous abortion. Prothrombin fragment 1 + 2, thrombin-antithrombin complex, and D-dimer transiently increased with dose; other coagulation parameters including PT, aPTT, ACT and platelet activity were unchanged.

Conclusions

PRT is a promising universal antidote for fXaI. A Phase 2 trial evaluating PRT reversal of several fXaI is ongoing.
Abstract 115: Microrna-30c Reduces Hyperlipidemia and Atherosclerosis by Decreasing Lipid Synthesis and Lipoprotein Secretion
Authors: Soh James , Iqbal Jahangir , Queiroz Joyce , Fernandez-Hernando Carlos , Hussain M. Mahmood
Published: July 2021

Hyperlipidemia is a risk factor for various cardiovascular and metabolic disorders. Overproduction of lipoproteins, a process critically dependent on microsomal triglyceride transfer protein (MTP), can contribute to hyperlipidemia. We have shown that hepatic over expression of miR-30c reduces MTP mRNA, protein and activity. Further, MTP mRNA is degraded faster due to the binding of miR-30c to its 3[[Unable to Display Character: ΄]]-UTR. miR-30c lowers plasma cholesterol by reducing production of triglyceride-rich apoB-containing lipoproteins; a phenotype most likely secondary to lower MTP expression. It also reduces
de novo
lipogenesis by targeting other genes such as LPGAT1. Additionally, atherosclerotic plaques are smaller in

Apoe
-/-

mice expressing miR-30c. Taken together, we have provided evidence that high miR-30c levels reduce plasma lipids and atherosclerosis and avoids steatosis by regulating different sets of genes.
Abstract 165: Saxagliptin Restores Vascular Mitochondrial Exercise Response in Diabetes
Authors: Keller Amy C , Knaub Leslie A , Miller Matthew W , Reusch Jane E
Published: July 2021

Exercise decreases cardiovascular disease (CVD) risk and all-cause mortality. We and others have defined that exercise stimulates mitochondrial biogenesis via upstream signaling through endothelial nitric oxide synthase (eNOS), sirtuins (SIRTs), and/or PPARγ co-activator alpha (PGC1-α). Further, this response is absent in diabetes and hypertension. We tested the hypothesis that pharmacological restoration of vascular signaling with the dipeptidyl peptidase-4 (DPP-4) inhibitor saxagliptin will improve vascular mitochondrial adaptation to exercise through activation of eNOS, SIRTs, and/or PGC1-α. We examined the impact of an 8 day treadmill exercise intervention in the Goto-Kakizaki (GK) rat, a model of lean, type 2 diabetes (T2DM), and Wistar control rats. Four groups were examined: sedentary or exercise groups with or without saxagliptin. Twenty-four hours post-bout, aortas were probed for mitochondrial complexes I-V, as well upstream signaling molecules. In Wistar rats, the expression of mitochondrial complexes III, IV, and V, along with SIRT3 (p<0.05), eNOS, and PCG1-α, increased with exercise. Conversely, in GK animals, exercise led to a decrease of complexes I, III, and IV, a decrease in the expression of COX IV (p<0.05) and AMPK, and no effect on eNOS or SIRT3. In exercised GK rats treated with saxagliptin, the expression of all mitochondrial complexes increased, complex IV significantly (p<0.05). Significant increases (p<0.05) were also observed in cytochrome c, eNOS and nNOS, PGC1-α, and UCP3 protein content in GK rats treated with both exercise and saxagliptin. Body weight and insulin and glucose concentrations were not significantly different in GK animals across experimental groups, indicating that the effects observed are independent of changes in these parameters. In addition, saxagliptin added to a three week exercise intervention improved running time significantly compared to an exercise intervention alone. In summary, our data suggest that saxagliptin restores vascular mitochondrial adaptation to exercise in a rodent model of diabetes. In conclusion, these data are proof of concept of a targetable mitochondrial defect in the diabetic vasculature.
Abstract 171: Acute Sagittal Sinus Thrombosis in an Elderly Patient with Chronic Idiopathic Thrombocytopenic Purpura
Authors: Wu Huimin , Paulino Robert , Shi Qi , Samavedam Sandhya , Pallekonda Indupriya , Khaira Rajni , Irukulla Pavan
Published: July 2021

INTRODUCTION

Chronic idiopathic thrombocytopenic purpura (ITP) is classified as a bleeding disorder that involves autoimmune platelet destruction. Cerebral venous sinus thrombosis in association with chronic ITP is rarely reported in medical literature.

CASE PRESENTATION

A 70-year-old female with a history of chronic ITP, on prednisone, and coronary artery disease presented with a four-day history of vomiting and diarrhea. Platelet count at the time of admission was 25×10
9
/L. She was treated with i.v normal saline. Platelets fell to 12×10
9
/L and intravenous immunoglobulin (IVIG) was started. Six days later, the patient developed confusion and right sided weakness. Repeat platelet count was 13x10
9
/L. MRI of the brain showed bilateral cortical infarcts suggesting sagittal vein involvement. MRV confirmed thrombosis of the posterior portion of the sagittal sinus. Bone marrow aspirates and core biopsy done to rule out other hematological disorders showed tri-lineage hematopoiesis with adequate marrow megakaryocytes. Hypercoagulable work up was unremarkable. ITP was treated with rituximab and romiplostim. Repeat MRV after 2 weeks showed no new thrombosis or hemorrhage.

DISCUSSION

ITP is occasionally associated with venous thrombosis. The paradoxical occurrence of venous thrombosis in the setting of ITP is poorly understood. Previous data postulates that platelet destruction releases humoral factors and microparticles, which may increase thrombotic events by the activation of thrombin and other coagulation factors. Other possible mechanisms include endothelial damage by autoantibodies directed against antigens on platelets and endothelial cells. More recently, reports have described IVIG-induced arterial and venous thrombotic complications. Treatment of thrombosis in the setting of ITP is complex and requires in-depth risk/benefit assessment. Our case highlights the dilemma of stroke treatment and prevention predicated on antiplatelet therapies for patients with ITP. Additional studies on chronic ITP associated with thrombosis are needed to establish preventive and treatment guidelines.
Abstract 190: Epigenetic Modifications of Pro-inflammatory Gene Expression in Macrophages by a Demethylase Enzyme, JMJD3, May Promote Chronic Inflammation in Type 2 Diabetic (T2D) Wounds
Authors: Gallagher Katherine A , Joshi Amrita , Carson William , Coleman Dawn , Henke Peter , Hogaboam Cory , Burant Charles , Kunkel Steven
Published: July 2021

Introduction

Type 2 diabetic(T2D) wounds are characterized by chronic inflammation, maintained by an exaggerated M1(pro-inflammatory) macrophage phenotype response. We seek to define a link between epigenetic modifications of bone marrow(BM) cells in T2D and dysregulated macrophages in wounds. We hypothesized that a chromatin modifying demethylase enzyme, JMJD3, is responsible for the decrease in H3K27me3 repressive methylation at the IL-12 gene promoter and thus drives an M1 macrophage phenotype in T2D wounds.

Methods

BM/adipose tissue(AT)/wounds were harvested from 30 diet-induced obese mice(DIO)(MG= 350g/DL) and 30 matched(WT) controls. For chromatin immunoprecipitation(ChIP) analysis, cells were isolated via ferromagnetic columns(CD34+,CD11b+). ChIP to detect histone methylation at the promoter regions of JMJD3 and IL-12(key M1 macrophage gene) was performed and RNA analysis was done with standard primers.

Results

JMJD3 mRNA in the BM is significantly increased in the DIO versus WT. ChIP showed increased H3K4me3(gene expression mark) in CD34+ progenitor cells and a corresponding decrease in H3K27me3(repressive mark) in monocytes at the promoter region of JMJD3. These changes correspond with the decrease in H3K27me3 seen at the IL-12 promoter in macrophages(CD11b+) from AT/T2D wounds.

Conclusions

Epigenetic changes initiated by JMJD3 in BM progenitor cells result in changes in histone methylation at the IL-12 promoter favoring an M1 phenotype in macrophages and thus contributes to the chronic inflammation seen in T2D wounds and AT. Whether manipulation of epigenetic enzymes could reduce chronic inflammation in T2D wounds requires further work.
Abstract 219: Agxt2 Plays Important Role in Regulation of Urine Adma Levels After Acute Adma Loading in Mice
Authors: Rodionov Roman N , Martens-Lobenhoffer Jens , Brilloff Silke , Hohenstein Bernd , Weiss Norbert , Bode-Böger Stefanie M
Published: July 2021

Background

Multiple epidemiological studies demonstrated increased levels of an endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) in cardiovascular diseases. In addition to the well characterized pathway of ADMA hydrolysis by dimethylarginine dimethylaminohydrolase (DDAH) ADMA can also be metabolized through an alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2), which converts ADMA to α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). The goal of this study was to better characterize the role of AGXT2 pathway in metabolism of ADMA
in vivo
using a recently developed assay for detection of DMGV in biological samples.

Methods

ADMA (250 μmol x kg-1 x d-1) was infused in C57/BL6 mice for 3 days using osmotic minipumps implanted intraperitoneally. We performed bilateral nephrectomy in some of the mice 24 hours before the end of ADMA infusion and sample collection. Urine was collected for 24 hours in metabolic cages. Blood was collected by cardiac puncture. Measurements of ADMA and DMGV were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results

Intraperitoneal infusion of ADMA for 3 days resulted in an increase in plasma ADMA levels from 0.46±0.045 to 1.57±0.40 μM (p<0.05) and in urine ADMA levels from 36.4±3.4 to 188.6±31.5 μmol / mmol creatinine (p<0.05). Elevation of ADMA concentration coincided with elevation of plasma DMGV levels from 0.21±0.025 to 0.61±0.14 μM (p<0.05) and urine DMGV levels from 41.4±3.2 to 162.5±32.9 μmol / mmol creatinine (p<0.05). Bilateral nephrectomy in the mice, which underwent ADMA infusion, lead to a 1.7 fold increase in plasma ADMA levels and a 17.6 fold increase in DMGV levels compared with the mice from the sham group.

Conclusions

Infusion of ADMA leads to increased flux through the AGXT2 pathway of ADMA metabolism in vivo. The observation that each 1 μmol / mmol creatinine increase in ADMA level in urine leads to a ~1 μmol / mmol creatinine increase in urine DMGV level suggests that AGXT2 is a major enzyme regulating ADMA levels in urine. The marked increase in DMGV levels in the mice lacking both kidneys suggests significant extrarenal production of DMGV in addition to absent renal excretion.
Abstract 236: Local Wall Thickness in Finite Element Models Improves Prediction of Abdominal Aortic Aneurysm Growth
Authors: Shang Eric K , Nathan Derek P , Fairman Ronald M , Woo Edward Y , Wang Grace J , Gorman Robert C , Gorman Joseph H , Jackson Benjamin M
Published: July 2021

Objective

Growing evidence suggests that peak wall stress (PWS) derived from finite element analysis (FEA) of abdominal aortic aneurysms (AAAs) predicts clinical outcomes better than diameter alone. Prior models assume uniform wall thickness (UWT). We hypothesize that inclusion of locally variable wall thickness (VWT) into FEA of AAAs will improve the ability to predict clinical outcomes.

Methods

Patients with AAAs (n=26) undergoing radiologic surveillance were identified. Custom MATLAB algorithms generated UWT and VWT aortic geometries from CTA images, which were subsequently loaded with systolic blood pressure using FEA. PWS and aneurysm growth (as a proxy for rupture risk and the need for repair) were examined.

Results

The average radiologic follow-up time was 22.0±13.6 months and the average aneurysm growth rate was 2.8±1.7 mm/year. PWS in VWT models significantly differed from PWS in UWT models (238±68 vs 212±73 kPa, P=0.025). In our sample, initial aortic diameter was not found to be correlated with aneurysm growth (r=0.26, P=0.19). A stronger correlation was found between aneurysm growth and PWS derived from VWT models as compared to PWS from UWT models (r=0.86 vs r=0.58, P=0.032 by Fisher’s r to Z transformation). The three panels in the figure demonstrate the correlation between aneurysm growth rate and (1) initial diameter, (2) PWS using UWT model, and (3) PWS using VWT model, respectively.

Conclusion

The inclusion of locally variable wall thickness significantly improved the correlation between PWS and aneurysm growth. Aortic wall thickness should be incorporated into future FEA models to accurately predict clinical outcomes.
Abstract 243: Adiponectin Inhibits Angiotensin II-induced Abdominal Aortic Aneurysm Formation
Authors: Vorkapic Emina , Wågsäter Dick , Van Stijn Caroline , Kim Jason , Eriksson Per , Tangirala Rajendra
Published: July 2021

BACKGROUND

Abdominal aortic aneurysms (AAA) are characterized by inflammation, loss of smooth muscle cells (SMC) and degradation of extracellular matrix in the vessel wall. Adiponectin, an adipokine produced by adipose tissue, exerts profound anti-inflammatory and anti-atherogenic effects on vascular cells. However, the role of adiponectin on AAA has not been studied. We investigated the impact of increased plasma adiponectin levels on angiotensin II (AngII)-induced AAA formation in mice.

METHOD AND RESULTS

Male low-density lipoprotein receptor-deficient (LDLR
-/-
) mice fed on a high-fat diet were infused with AngII (25 mg/kg/min) for 8 weeks to induce AAA. Mice infused with saline was used as controls. A recombinant adenoviral vector encoding mouse adiponectin (Ad-APN) was injected intravenously to induce adiponectin expression. A recombinant adenovirus expressing green fluorescence protein (GFP) was used as a control (Ad-GFP). Eight weeks after injection, plasma adiponectin levels were 10-fold higher (253.9±59.0 μg/mL) in Ad-APN mice compared to Ad-GFP mice (25.2±3.8 μg/mL, P<0.001). Aortic diameter was significantly reduced in Ad-APN mice compared to Ad-GFP mice (60% reduction). This was accompanied by decreased frequency and size of abdominal atherosclerotic lesions in Ad-APN mice compared to Ad-GFP mice. Histologically, elastin was degraded in all AngII-infused mice compared to saline-infused mice. However elastin was more preserved in Ad-APN mice compared to Ad-GFP mice (P<0.05). Gene expression analysis of the abdominal aorta revealed a decrease of CD3e (P<0.05) but no profound changes in inflammatory genes, IL-6, TNF-α and CD68. Decreased mRNA expression of the SMC marker SM22α was observed in all AngII-infused mice compared to saline-infused mice. Expression of SM22α was two-fold higher in the abdominal aorta in Ad-APN mice than in Ad-GFP mice (P<0.05).

CONCLUSION

These results provide first evidence that adiponectin overexpression significantly inhibits AngII-induced AAA formation in LDLR
-/-
mice. The protective actions of adiponectin appear to be mediated, in part, through preservation of SMC content and elastin in the vascular wall. The mechanisms of adiponectin inhibiting AAA formation deserve further attention.
Abstract 249: Delayed Gene Transfer Increases Transgene Expression in Vein Grafts
Authors: Du Liang , Zhang Jingwan , Clowes Alexander , Dichek David
Published: July 2021

Background

Autogenous vein grafts are effective therapies for obstructive arterial disease. However, their long-term utility is limited by stenosis and occlusion. Genetic engineering of veins that prevents intimal hyperplasia and atherosclerosis could significantly improve the clinical utility of vein grafts. We recently reported that a helper-dependent adenoviral vector (HDAd) reduces atherosclerosis 4 wks after gene transfer in fat-fed rabbits and can express a therapeutic transgene (apo AI) in normal rabbit carotids for at least 48 wks. Use of HDAd for vein graft gene therapy will depend on achievement of similarly high and persistent transgene expression in grafted veins.

Hypothesis

We tested the hypothesis that Ad-mediated transgene expression in grafted veins (at an early time point) can be increased by varying the timing of gene transfer.

Methods

Rabbit external jugular veins were transduced by exposure to a beta galactosidase (b-gal)-expressing Ad: in situ either without (a) or with (b) immediate arterial grafting; c) ex vivo with grafting after overnight incubation with Ad; d) in vivo immediately after grafting and e) in vivo 4 wks after grafting (n = 6 - 19 veins/group). Transgene expression was measured in veins removed 3 d after Ad exposure by PCR quantitation of b-gal mRNA and by en-face planimetry of blue-stained area.

Results

B-gal transgene expression was higher in ungrafted veins than in veins grafted immediately after gene transfer (84 ± 17 vs 9.4 ± 2.0 arbitrary units (AU); P < 0.0001). Overnight incubation of veins with Ad increased gene expression ex vivo by 10-fold but neither this nor performing vector infusion immediately after grafting improved gene expression (11 ± 4.7 and 9.1 ± 1.8 AU; P > 0.9 for both vs immediately grafted veins). Delaying gene transfer until 4 wks after grafting significantly increased gene expression, to a level equivalent to transgene expression in ungrafted veins (61 ± 11 AU; P = 0.3 vs ungrafted veins). En face planimetry yielded similar results.

Conclusions

Exposure of a transduced vein to arterial blood flow is associated with significant loss of transgene expression. Transgene expression in grafted veins is significantly higher when gene transfer is performed 4 wks after exposure of the vein to arterial blood flow.
Abstract 286: Loss of Smad3 Exacerbates Abdominal Aortic Aneurysm Formation With Enhanced Inflammation in an Experimental Mouse Model
Authors: Dai Xiaohua , Arora Anandita , Shen Jianbin , Jiang Hong , Li Li
Published: July 2021

Introduction

Abdominal aortic aneurysm (AAA) is a complex vascular disease that causes more than 10,000 deaths each year in the United States. Extensive studies have been performed in search of pharmaceutical treatment but surgical repair still remains the most effective treatment. TGF-β signaling is an important mechanism in the pathogenesis of aneurysms; however, there is debate as to whether its role is protective or destructive. Smad3 is a major intracellular mediator of the canonical pathway of TGF-β signaling.

Hypothesis

We hypothesize that Smad3-mediated TGF-β signal pathway plays important roles in the pathogenesis of AAA.

Methods

To test this hypothesis, we analyze the effects of loss of Smad3 on aneurysm formation in the calcium chloride induced AAA model using Smad3 knockout mice.

Results

Three weeks after calcium chloride treatment, the abdominal aorta displayed increased dilation, forming aneurysms. Histology and immunohistochemistry analyses show increased cell proliferation and enhanced inflammatory cell infiltration in the media and adventitia of the vessel wall. This was accompanied by elastic fibers degradation, increased MMPs expression and reduced expression of smooth muscle markers. Further analysis showed that the expression and nuclear localization of Smad2 and Smad4 was significantly increased.

Conclusions

These results demonstrate that Smad3-mediated TGF-β signaling plays a protective role in the pathogenesis of AAA and Smad2/Smad4 upregulation is not sufficient to compensate for the loss of Smad3 in this experimental model.
Abstract 289: High Density Lipoproteins Augment Hypoxia-Induced Angiogenesis via Regulation of Post-translational Modification of HIF-1a
Authors: Tan Joanne T , Prosser Hamish C , Vanags Laura Z , Monger Steven A , Ng Martin K , Bursill Christina A
Published: July 2021

Background

Angiogenesis plays a critical role in tissue neovascularization in response to ischemia, particularly following myocardial infarction (MI). High-density lipoproteins (HDL) are associated with improved survival following MI, suggesting that it may play a role in hypoxia-induced angiogenic regulation. We have shown previously that in hypoxia, HDL augments angiogenesis and HIF-1α/VEGF expression. HIF-1α is post-translationally regulated by prolyl hydroxylases (PHDs 1-3), which target HIF-1α for degradation following hydroxylation. Furthermore, Siah ubiquitin ligases (Siah1 and Siah2) promote the degradation of PHDs. We aimed to determine if HDL augments HIF-1α/VEGF via modulation of PHDs and Siahs.

Methods

Human coronary artery endothelial cells were pre-incubated with PBS (control) or reconstituted HDL (rHDL) for 24 h then subjected to normoxic or hypoxic conditions for 6 h. To determine the importance of Siah1 and Siah2 in the HDL-induced modulation of the HIF-1α/VEGF pathway, a siRNA knockdown approach was used.

Results

Pre-incubation with rHDL inhibited the hypoxia-induced increases in PHD2 and PHD3 levels (32% and 45% respectively, p<0.05) while Siah1 and Siah2 mRNA levels were increased (58% and 88% respectively, p<0.05) with rHDL pre-incubation. Following siRNA knockdown of Siah1 and Siah2, HDL lost its ability to induce the expression of HIF-1α (11% reduction, p<0.05) and VEGF (18% reduction, p<0.05). Augmentation of tubulogenesis by HDL was attenuated in Siah siRNA-transfected cells (55% in siSiah1; 40% in siSiah2, p<0.05). Siah knockdown also abrogated HDL inhibition of hypoxia-mediated induction of PHD2 and PHD3 (p<0.05). PI3K and Akt are upstream regulators of HIF-1α/VEGF and also regulate Siahs. Specific inhibition of the PI3K/Akt pathway revealed that it plays a key role in HDL-induced elevations in Siah1 and Siah2 (37% and 20% reduction respectively, p<0.05) as well as HIF-1α and VEGF (65% and 15% reduction respectively, p<0.05).

Conclusion

In conclusion, HDL augments the HIF-1α/VEGF pathway by regulation of post-translational modification of HIF-1α via the PI3K/Siah/PHD pathway. HDL induced augmentation of angiogenesis in hypoxia may have implications for preventing ischemic injury following MI.
Abstract 3: Apolipoprotein A-I Inhibits Streptococcal Cell Wall-Induced Arthritis in the Rat in an ABCA1-dependent Manner
Authors: Wu Ben J , Ong Kwok L , Shrestha Sudichhya , Chen Kang , Barter Philip J , Rye Kerry-Anne
Published: July 2021

Introduction.

Arthritis is a chronic inflammatory disease characterized by joint inflammation and destruction, reduced high-density lipoprotein (HDL) levels, and increased cardiovascular risk.

Objective

To determine if apolipoprotein (apo) A-I, the main HDL apolipoprotein, prevents joint inflammation in arthritis.

Methods and Results

In vivo: Arthritis was induced in female Lewis rats with a single 15 mg/kg intraperitoneal streptococcal cell wall peptidoglycan-polysaccharide (PG-PS) injection and quantified as a combined forepaw and hindpaw inflammation score. Arthritis progressed from an initial, acute phase of joint inflammation during the first 4 days post-PG-PS administration to remission by day 8, followed by chronic joint inflammation up to sacrifice at day 21. Two intravenous infusions of lipid-free apoA-I (8 mg/kg) 24 h pre- and 24 h post-PG-PS injection reduced the acute and chronic joint inflammation by 63±9% at day 3 and by 61±8% at day 21. Infusion of apoA-I at days 7, 9 and 11 post-PG-PS injection reduced the chronic response by 43±11% at day 21. ApoA-I infusions at 24 h prior to and at days 1, 7, 9, 11 post-PG-PS injection reduced joint inflammation by 61±5% at day 3 and by 90±5% at day 21 (p<0.05 for all vs saline infusion). These beneficial effects of apoA-I were accompanied by a reduced inflammatory white blood cell count, reduced pro-inflammatory cytokine levels in synovial fluid, and reduced macrophage accumulation, toll-like receptor 2 (TLR2) and inflammatory cytokine expression in synovial tissue. In vitro: Human monocyte-derived macrophages (HMDMs) were pre-incubated with lipid-free apoA-I, then stimulated with PG-PS (20 μg/mL). Pre-incubation with apoA-I inhibited PG-PS-induced TLR2 and MyD88, a TLR2 adapter protein, expression. Nuclear factor-κB activation and pro-inflammatory cytokine production were also attenuated. These anti-inflammatory effects of apoA-I were abolished in HMDMs transfected with ATP-binding cassette transporter 1 (ABCA1) siRNA.

Conclusions

These findings establish that apoA-I attenuates PG-PS induced arthritis in the rat. These effects may involve ABCA-1 and inhibition of TLR2 expression and activation.
Abstract 335: Vascular Histone Deacetylase Mediates Early Life Stress-Induced Endothelial Dysfunction
Authors: Ho Dao H , Pollock Jennifer S
Published: July 2021

Chromatin remodeling is an important factor in the etiology of vascular pathologies. Also, early life stress (ELS) is linked to increased risk of vascular disease in adults. We used maternal separation with early weaning (MSEW) to study mechanisms of ELS-mediated adult vascular dysfunction in male C57BL/6J mice. Litters were subjected to maternal separation 4h/day (postnatal day (PD) 2-5) and 8h/day (PD6-16), and weaned at PD17. Control (CON) litters were undisturbed until weaning at PD21. Subsequent experiments were performed at 12 weeks old. MSEW blunted aortic ACh-mediated vasorelaxation (MSEW: 68% vs CON: 90%, p=0.01), while SNP-induced vasorelaxation was similar in CON and MSEW aortae. Apocynin (300 μM) and superoxide dismutase (100 U/mL) normalized MSEW-induced endothelial dysfunction. We hypothesize that ELS induces aortic endothelial dysfunction by increasing NADPH oxidase expression and/or decreasing nitric oxide synthase 3 (NOS3) expression. Aortic protein expression of NADPH oxidase subunit p67 was elevated in MSEW mice (45% increase from CON, n=11, p=0.02). NOS3 protein expression and NOS3 serine 1177 phosphorylation was not different between groups, indicating that NOS3 activation by phosphorylation does not contribute to ELS-induced endothelial dysfunction. We further hypothesize that chromatin modification mediates ELS-induced endothelial dysfunction. Aortic mRNA expressions of 84 chromatin modification enzymes (methyltransferases, demethylases, acetyltransferases, deacetylases) were assessed by qRT-PCR. Only histone deacetylase (HDAC) 1, 6 and 9 mRNA levels were significantly upregulated in MSEW aortae compared to CON (17%, 29% and 67% increase, respectively, p<0.05). However, only HDAC 9 protein expression was elevated in MSEW aortae (2 fold increase from CON, n=6, p=0.01). Accordingly, histone 3 lysine acetylation was slightly decreased in MSEW aortae (16% decrease from CON, n=6, p = 0.06). Pretreatment of aortae with an HDAC inhibitor, trichostatin A (TSA), normalized ACh-induced vasorelaxation in MSEW mice (MSEW: 68% vs MSEW + TSA: 88%, p=0.02), while not affecting ACh-induced vasorelaxation in CON mice. We conclude that ELS induces endothelial dysfunction, most likely, through an HDAC 9-mediated pathway.
Abstract 357: Hepatic ERK2 Suppresses Endoplasmic Reticulum Stress, Leading to Protection from Oxidative Stress and Endothelial Dysfunction
Authors: Kujiraoka Takehiko , Satoh Yasushi , Ayaori Makoto , Shiraishi Yasunaga , Arai-Nakaya Yuko , Hakuno Daihiko , Yada Hirotaka , Endo Shogo , Isoda Kikuo , Adachi Takeshi
Published: July 2021

Background

Insulin signaling comprises 2 major cascades, the IRS/PI3K/Akt and Ras/Raf/MEK/ERK pathways. Many studies on the tissue-specific effects of the former pathway had been conducted, however, the role of the latter cascade in tissue-specific insulin resistance had not been investigated. High glucose/fatty acid toxicity, inflammation and oxidative stress, all of which are associated with insulin resistance, can activate ERK. Liver plays a central role of metabolism and hepatosteatosis (HST) is associated with vascular diseases. The aim of this study is to elucidate the role of hepatic ERK2 in HST, metabolic remodeling and endothelial dysfunction.

Methods

Serum biomarkers of vascular complications in human were compared between subjects with and without HST diagnosed by echography for regular medical checkup. Next, we created liver-specific ERK2 knockout mice (LE2KO) and fed them with a high-fat/high-sucrose diet (HFHSD) for 20 weeks. The histological analysis, the expression of hepatic sarco/endoplasmic reticulum (ER) Ca
2+
-ATPase 2 (SERCA2) and glucose-tolerance/insulin-sensitivity (GT/IS) were tested. Vascular superoxide production and endothelial function were evaluated with dihydroethidium staining and isometric tension measurement of aorta.

Results

The presence of HST significantly increased HOMA-IR, an indicator of insulin resistance or atherosclerotic index in human. HFHSD-fed LE2KO revealed a marked exacerbation in HST and metabolic remodeling represented by the impairment of GT/IS, elevated serum free fatty acid and hyperhomocysteinemia without changes in body weight, blood pressure and serum cholesterol/triglyceride levels. In the HFHSD-fed LE2KO, mRNA and protein expressions of hepatic SERCA2 were significantly decreased, which resulted in hepatic ER stress. Induction of vascular superoxide production and remarkable endothelial dysfunction were also observed in them.

Conclusions

Hepatic ERK2 revealed the suppression of hepatic ER stress and HST
in vivo
, which resulted in protection from vascular oxidative stress and endothelial dysfunction. HST with hepatic ER stress can be a prominent risk of vascular complications by metabolic remodeling and oxidative stress in obese-related diseases.