bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 163: Functional Analysis of Novel Mutations in Human SR-BI
Authors: Chadwick Alexandra C , Sahoo Daisy
Published: March 2022

Scavenger receptor class B, type I (SR-BI) is an 82 kDa glycosylated high density lipoprotein (HDL) receptor that is imperative for mediating HDL-cholesterol (HDL-C) uptake into the liver for whole body cholesterol excretion. Recently, two novel point mutations in human SR-BI, S112F or T175A, were discovered in patients with high HDL-C levels. We hypothesize that the high HDL-cholesterol levels are a direct result of each mutant SR-BI’s inability to bind and mediate the selective uptake of cholesteryl esters (CE) from HDL particles. To test this hypothesis, we created SR-BI cDNA encoding the S112F- or T175A point mutations. Cell surface biotinylation and flow cytometry analysis confirmed the plasma membrane localization of both mutant receptors, although T175A-SR-BI expressed at slightly lower levels than wild-type SR-BI. Following transient transfection into COS-7 cells, both S112F- and T175A-SR-BI mutant receptors displayed a greater than 75% decrease in [125I]HDL binding and a greater than 90% decrease in selective uptake of [3H]HDL-COE, as compared to wild-type SR-BI. Both mutant SR-BI receptors also displayed an impaired ability to mediate the release of free cholesterol from cells to HDL. PFO-PAGE revealed that the mutant receptors maintained their ability to form dimers and higher order oligomers, suggesting that the lack of function is not due to a change in the oligomeric status of SR-BI. Interestingly, introduction of alanine at residue 175 disrupts the consensus sequence for N-linked glycosylation at Asn173. Immunoblot analysis confirmed that the T175A-SR-BI migrated slightly faster than wild-type SR-BI, and we hypothesize that receptor function may be compromised due to changes in the glycosylation pattern of this receptor. It is also important to note that S112 and T175 are located in a highly conserved hydrophobic region of the extracellular domain that has previously been shown to be important for receptor function. As such, we speculate that mutations at one or both of these residues may also have a significant impact on HDL-cholesterol transport due to changes in hydrophobicity of this region of SR-BI.
Abstract 164: The Chaperone Holdase Bag6 Modulates the Intracellular Fate of Newly Synthesized ApoB100
Authors: Fisher Eric A , McLeod Roger S
Published: March 2022

Apolipoprotein B-100 (apoB) is the central protein component of very low density lipoproteins (VLDL) and is subject to endoplasmic reticulum-associated degradation (ERAD). Hepatic assembly of VLDL particles is monitored by the cellular quality control machinery, which determines whether a properly formed lipoprotein is secreted or the inadequately lipidated apoB protein is degraded. Changes in lipid supply, ER folding capacity or ERAD pathway function can alter VLDL output from HepG2 cells. Bag6 is a cytosolic protein that has a role in the bilayer integration of proteins and is also implicated in maintaining the solubility of proteasome substrates that contain exposed hydrophobic regions. Notably, Bag6 has been observed to interact with the ERAD components gp78 and p97, which are known to facilitate apoB degradation. The goal of this study was to evaluate the role of cytosolic Bag6 in apoB metabolism in HepG2 cells. When MG132 was used to inhibit proteasomal degradation of apoB, Bag6 was co-immunoprecipitated with apoB as determined by functional proteomics and immunoblot analysis. Conversely, if VLDL assembly was enhanced by treatment of the cells with the MEK/ERK inhibitor U0126, both the Bag6-apoB and p97-apoB associations were no longer detectable. Metabolic labelling studies indicated that siRNA knockdown of Bag6 did not alter the initial rate of apoB biosynthesis, but decreased the secretion of newly synthesized apoB. The decrease in apoB secretion with Bag6 knockdown could not be reversed by U0126. In addition, Bag6 knockdown decreased the effectiveness of MG132 as an inhibitor of cellular apoB degradation even though ubiquitination of apoB appeared to be unaffected. These observations suggest that Bag6 may be a component of the ERAD pathway that modulates VLDL assembly. Reduction of Bag6 partially decreases both secretion and MG132-inhibitable degradation of apoB, suggesting that another mechanism is also involved in apoB degradation. We hypothesize that the “holdase” function of Bag6 may temporarily stabilize exposed hydrophobic domains of apoB prior to its progression to assembly or degradation.
Abstract 165: RNAi Therapeutics Targeting PCSK9 and ANGPTL3 for Mixed Hyperlipidemia
Authors: Frank-Kamenetsky Maria , Shulga-Morskaya Svetlana , Liebow Abigail , Racie Tim , Milstein Stuart , Kuchimanchi Satya , Charisse Klaus , Epstein-Barash Hila , Fitzgerald Kevin
Published: March 2022

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase (PC) family of subtilisin-like serine endoproteases that regulates low density lipoprotein receptor (LDLR) levels and function. Loss of PCSK9 protein (in murine models, as well as, in human individuals) increases LDLR levels while excess PCSK9 decreases LDLR levels. These changes in LDLR protein levels coincide with reciprocal changes in circulating levels of plasma LDL cholesterol. Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted proteins. Similar to PCSK9, it is predominantly expressed in the liver. ANGPTL3 acts as dual inhibitor of both lipoprotein and endothelial lipases. Animal studies as well as human genetic studies suggest that siRNA silencing of PCSK9 should result in substantial decrease in LDLc. Similar studies have suggested that liver silencing of ANGPTL3 should result in profound lowering of LDLc, Triglycerides and HDLc, while maintaining the HDLc/LDLc ratio. We have developed highly potent RNAi therapeutics targeting both PCSK9 and ANGPTL3. Pre-clinical data utilizing intravenous infusion of siRNA formulated in lipid nanoparticles, and subcutaneous delivery of siRNA-GalNAc conjugates will be discussed.
Abstract 240: High-Density Lipoproteins Augment Hypoxia-Induced Angiogenic Regulation via Mediators in the HIF-1α Signaling Pathway
Authors: Tan Joanne , Prosser Hamish , Vanags Laura , Ng Martin , Bursill Christina
Published: March 2022

Background:
Angiogenesis plays a critical role in tissue neovascularization in response to ischemia, particularly in collateral vessel development following myocardial infarction (MI). One of the key pathways involved in hypoxia-induced angiogenesis is the HIF-1α/VEGF pathway. High-density lipoproteins (HDL) are associated with improved survival and prognosis following MI, suggesting that it may play a role in hypoxia-induced angiogenic regulation. We assessed the hypothesis that HDL regulates the HIF-1α/VEGF pathway
in vitro
.

Methods:
Human coronary artery endothelial cells (HCAECs) were pre-incubated with PBS (control) or reconstituted HDL (rHDL) for 24 hrs then subjected to either normoxic or hypoxic conditions for a further 6 hrs. Protein and gene expression of key mediators in the HIF-1α/VEGF pathway were investigated.

Results:
As expected, hypoxia elevated HIF-1α protein levels (49%, p<0.05), which was augmented by a further 30% (p<0.05) in rHDL-treated HCAECs. Consistent with this, rHDL increased VEGF mRNA levels above hypoxia-induced increases (23%, p<0.05). Prolyl hydroxylases (PHD 1-3) hydroxylate HIF-1α, targeting it for degradation. rHDL pre-incubation inhibited hypoxia-induced increases in PHD2 and PHD3 levels (32% and 45%, respectively, p<0.05), indicating that rHDL may protect against HIF-1α degradation and explains the rHDL-induced elevation in HIF-1α protein levels. Furthermore, the mRNA levels of Siah-1 and Siah-2, which promote PHD degradation, were increased in rHDL-treated HCAECs (58% and 88% respectively, p<0.05). To determine the importance of Siah-1 and Siah-2 in the HDL-induced modulation of the HIF-1α/VEGF pathway, a siRNA knockdown approach was used. When cells had low levels of Siah-1 and -2, HDL’s ability to induce VEGF was completely attenuated.

Conclusion:
In conclusion, we found that HDL is able to augment the HIF-1α/VEGF pathway through the stabilization of HIF-1α protein via a decrease in PHD2/3 and increase in Siah1/2. These findings suggest that rHDL may stimulate hypoxia-induced angiogenic regulation via the HIF-1α pathway, which may have implications for preventing ischemic injury following MI.
Abstract 265: Morning Dosing of Eplerenone Improves Daytime Blood Pressure Control but Not Fibrinolytic Balance
Authors: Muldowney James A , Schoenhard John A , Vaughan Douglas E
Published: March 2022

The renin angiotensin-aldosterone system has a diurnal variation, proceeding morning increases in blood pressure and plasminogen activator inhibitor-1 (PAI-1) levels. The RAAS is particularly active in individuals with metabolic syndrome (MetS) resulting in increased blood pressure and elevated PAI-1 levels. Eplerenone is a mineralocorticoid antagonist that is approved for the treatment of hypertension with a half-life of 4-6 hours but can be prescribed once or twice daily. This is a double-blind crossover study that examines the effects of morning (AM) vs. nighttime (PM) administration of eplerenone on blood pressure, PAI-1, plasma renin activity (PRA), and plasma aldosterone levels. Twenty subjects with MetS were studied on three 24-hour inpatient study days on hydrochlorothiazide (25mg) alone and with concomitant morning or evening dosing of eplerenone (100mg). Vital signes were taken and blood was drawn every three hours. There was a statistically significant increase in aldosterone levels with both AM (266±21 ng/dL) and PM (320±31 ng/dL) dosing of eplerenone compared to baseline (194±11 ng/dL) (both p < 0.001) with no significant difference between the two dosing strategies. There was a trend suggesting a daytime (8AM-5PM) systolic blood pressure reduction of 4.9±1.4 mmHg (p = 0.06) with AM dosing. Furthermore, diastolic blood pressure appeared less volatile with AM dosing (range = 38 mmHg vs. 47 mmHg for baseline and PM). There was no difference between baseline, AM and PM dosing with regard to PRA, PAI-1, serum potassium or creatinine. These findings suggest that there may be improved overall control of blood pressure with AM dosing of eplerenone, without obvious deleterious effects such as elevated creatinine or potassium levels. Furthermore, increases in aldosterone did not result in increases in PAI-1. Consequently rational dosing strategies such as this merit further investigation.
Abstract 280: Evidence for the Beneficial Effects of Ixmyelocel-T in the Treatment of Critical Limb Ischemia
Authors: Ledford Kelly , Zeigler Frank , Bartel Ronnda
Published: March 2022

Ixmyelocel-T, an expanded, autologous, multicellular therapy cultured from bone marrow, has recently demonstrated clinical promise in the treatment of patients with critical limb ischemia. At this time, mechanisms describing its beneficial effects remain unclear. We investigated the potential mechanisms by which ixmyelocel-T may improve recovery from ischemia. In a rat model of hind limb ischemia, ixmyelocel-T treated rats exhibited significantly increased blood flow recovery by comparison to their vehicle control counterparts, as measured by laser Doppler. Ixmyelocel-T treated rats exhibited increased plasma nitrate levels (6.2±2.8 vs 32.8±12.4 μM, p < 0.05) measured with the Greiss reaction, and decreased plasma lipid peroxidation (0.04±0.005 vs.0.03±0.003 μM, p < 0.05) measured with the thiobarbituric acid reactive substances (TBARS) assay, indicating a preservation in the bioavability of nitric oxide. Fluorescent imaging revealed that PKH-labeled ixmyelocel-T co-localizes with eNOS in the blood vessels of the ischemic limb. Co-culture experiments with human umbilical vein endothelial cells (HUVEC) were employed to determine the effect of ixmyelocel-T on the endothelium. Co-culture resulted in increase of eNOS (1516±225 vs.1995±167 pg/ml, p < 0.05), as measured by ELISA, and nitric oxide (NO) production (71±1.4 vs.75±1.3 AU, p < 0.05), as measured with the intracellular NO probe DAF-2DA. Co-culture also resulted in reduced reactive oxygen species in TNFα activated HUVECs measured by an intracellular ROS probe (4.8±1.6 vs.1.4±0.4 nM DCF), as well as increased the release of the anti-inflammatory cytokine IL-10 by ixmyelocel-T. Ixmyelocel-T also promoted HUVEC migration in an
in vitro
scratch assay. Collectively, our data suggests that ixmyelocel-T stimulates recovery from ischemia through mechanisms involving stimulation of eNOS expression, increasing nitric oxide production, reducing oxidative stress and inflammation, and promoting endothelial migration, thereby promoting the preservation of vascular function. We hypothesize that these functions of ixmyelocel-T are responsible for the recovery of ischemic tissues in patients with CLI.
Abstract 285: The Hemodynamic Actions of Insulin Are Blunted in the Sublesion Microvasculature of Healthy Persons with Spinal Cord Injury
Authors: La Fountaine Michael F , Rosado-Rivera Dwindally , Radulovic Miroslav , Bauman William A
Published: March 2022

Background:
The hemodynamic actions of insulin (HAI) involve endothelial-mediated and sympathetic nervous system (SNS)-facilitated vasodilatation of the microvasculature. Spinal cord injury (SCI) is characterized by interruption of supraspinal SNS control of sub-lesion vasomotor tone, and a high prevalence of disordered carbohydrate metabolism, cardiovascular-related morbidity and pressure ulcers, are well appreciated.

Objective:
To gain a better understanding of the HAI on sub-lesion microcirculation in SCI.

Procedures:
Insulin iontophoresis (InsI; FDA IND #110886) was performed in an otherwise healthy cohort of persons with SCI and an age- and gender-matched cohort of neurologically intact control subjects. Laser Doppler flowmetry characterized peak blood perfusion unit (BPU) responses (%change from baseline) to InsI or placebo iontophoresis (PlacI) above and below the neurological level of injury. Parametric statistics were employed to test for statistical differences within and between condition (InsI, PlacI), location (arm, leg), and group (SCI, control). An a priori level of significance was set at p≤0.05.

Results:
Demographics (SCI: n=10, age 39 ± 12, 8 males: control: n=10, age 37 ± 10, 8 males), fasting plasma glucose (SCI: 5.2 ± 0.5; control 5.3±0.2 mmol/l) and fasting plasma insulin (SCI: 12.4 ± 7.8; control 13.1 ± 5.8 μIU/ml) concentrations did not differ between groups. PlacI did not result in any significant BPU changes in the arm or leg of either group. In the arm, insulin iontophoresis resulted in a 20% average BPU increase (p<0.05) in controls and a 9% increase in the SCI group (p=0.14). In the legs, InsI resulted in an 81% (p<0.01) and 29% (p<0.001) average BPU increase in the control and SCI groups, respectively, a change that was significant between groups (p<0.05). InsI of the leg resulted in a 19% (p<0.05) and 61% (p<0.05) greater BPU increase than the arm in the SCI and control groups, respectively.

Conclusion:
The HAI are blunted in the sub-lesion microvasculature of persons with SCI. Our findings could have implications for the prevalence and genesis of skin breakdown, the initiation of pressure ulcers and/or cardiovascular disease and peripheral insulin insensitivity due to reduced tissue perfusion in persons with SCI.
Abstract 292: ETC-1002 Reduces Body Weight Gain and Hepatic Triglyceride Content and Improves Glycemic Control in a Mouse Model of Diet-Induced Obesity
Authors: Hanselman Jeffrey C , Srivastava Rai A , Hurley Timothy R , Filippov Sergey , Spahr Mark A , Bradshaw Cheryl D , Cramer Clay T , Lister Richard J , Pinkosky Stephen L , Newton Roger S
Published: March 2022

ETC-1002 is an investigational drug currently in Phase 2 clinical development to treat dyslipidemia and other cardiometabolic risk factors. Previously, ETC-1002 prevented hyperlipidemia and atherosclerosis in rodent models; and improved hepatic triglycerides (TG) as well as fasting blood glucose and insulin in the KKA
y
insulin resistant mouse model via putative mechanisms including activation of AMP-activated protein kinase. In the present study we investigated the effect of ETC-1002 on body weight, hepatic TG and insulin sensitivity in a diet-induced obese (DIO) mouse model. C57BL/6 mice were fed a 60% high-fat diet beginning at 11 weeks of age. At 12 weeks of age mice were assigned to treatment groups and administered vehicle or ETC-1002 at 3, 10, or 30 mg/kg/day for 9 weeks. A separate cohort of mice was maintained on standard rodent chow diet throughout the study as a comparator. Food consumption, body weight, hepatic TG content, fasting blood glucose, fasting plasma insulin, insulin tolerance tests, and glucose tolerance tests were measured. Mice developed obesity, hyperinsulinemia, mild hyperglycemia and elevated hepatic triglycerides in response to the high-fat diet. ETC-1002 results were dose-dependent and statistically significant at doses of 10 and 30 mg/kg/day. ETC-1002 attenuated body weight gain 8% and 15% with no effect on food consumption. Body weight changes were associated with 12% and 32% decreases in epididymal fat pad mass. Hepatic TG content was also reduced with ETC-1002 by 34% and 46%; respectively. ETC-1002 treatment reduced fasting blood glucose 11% and 16%; plasma insulin 80% and 95%; and resulted in significant improvements in insulin tolerance tests (19% and 22% reduction in AUC) with modestly improved glucose tolerance (not significant). In an intervention study with a 12 week lead-in on high-fat diet, comparable effects on body weight, hepatic TG, and insulin sensitivity were observed. In summary, ETC-1002 reduced obesity and hepatic TG and improved glycemic parameters in a high-fat fed diet-induced mouse model of disease. The present data in the DIO mouse, combined with previously reported efficacy in rodent models supports ETC-1002 as a regulator of imbalances in lipid and carbohydrate metabolism.
Abstract 298: Vitamin D3 Supplementation in Obesity Corrects the Renal Vascular Response to Angiotensin II Akin to the Action of a Converting Enzyme Inhibitor
Authors: Vaidya Anand , Sun Bei , Larson Carol , Williams Jonathan
Published: March 2022

Background:
Increased activity of the renal-vascular renin-angiotensin system (RAS) is a leading mechanism for chronic kidney disease (CKD) in obesity. Low vitamin D levels are associated with increased RAS activity, obesity, and the development of CKD; we hypothesized that vitamin D3 therapy in obesity would lower renal-vascular RAS activity and thus increase the renal-vascular sensitivity to angiotensin II (AngII) akin to an ACE inhibitor.

Methods:
14 morbidly obese subjects (BMI 36 kg/m2) with hypertension, pre-diabetes, 25(OH)D<25 ng/mL, and normal renal function were studied. Anti-hypertensive medications were withdrawn for up to 3 months, and subjects completed a controlled sodium diet for 1 week prior to study visits, which occurred before and after 15,000 IU/daily of vitamin D3 for 1 month. At each study visit, renal plasma flow (RPF, via p-aminohippurate clearance) was measured during an infusion of AngII (1 ng/kg/min) in triplicate at baseline, 45, and 90 mins. Subjects were then treated with captopril and underwent a second infusion of AngII to evaluate the change in renal-vascular RAS induced by captopril. Repeated-measures regression analyses were used to examine the effect of vitamin D3 therapy on RPF while accounting for variation in PAH measurements and the lack of independence between intra-individual observations.

Results:
With vitamin D3 therapy, mean 25(OH)D rose from 18 to 52 ng/mL, PTH fell from 46 to 37 pg/mL, and baseline RPF rose from 383.6 to 401.0 mL/min/1.73m2 (P<0.01). Vitamin D3 intervention modified the influence of AngII infusion on RPF; there was a greater decline in RPF (P<0.05) following vitamin D3 therapy, suggesting a reduction in renal-vascular RAS. After a dose of captopril, the renal-vascular sensitivity to AngII was significantly augmented at both study visits, but this improvement was much smaller following vitamin D3 therapy (P<0.05); raising vitamin D levels corrected the renal-vascular sensitivity to AngII similarly to ACE inhibition.

Conclusions:
In this controlled physiology study, vitamin D3 therapy in obese hypertensives increased basal RPF and the renal-vascular sensitivity to AngII akin to the effect of an ACE inhibitor. Chronic vitamin D3 therapy in obesity may reduce the risk of CKD by reducing RAS activity.
Abstract 312: Frequencies and Functional Characterization of Regulatory T Cell Subsets in Patients with Coronary Artery Disease
Authors: Hasib Lekbira , Jonasson Lena , Backteman Karin , Ernerudh Jan
Published: March 2022

Background:
There is evidence for a protective role of regulatory T cells (Tregs) in atherosclerosis. A few studies have analysed Tregs in patients with coronary artery disease (CAD) although with contradictory results. Our aim was to characterize naïve(n) Tregs and memory(m) Tregs in patients with CAD.

Methods:
Phenotypic analysis of circulating Tregs (including activation markers CTLA4 and Helios) was performed in 21 patients with CAD (all statin-treated) and 24 controls by flow cytometry. CD4+ T cells were enriched by negative selection followed by positive selection using anti-CD4, anti-CD25, anti-CD127, and anti-CD45RA. The fractions of CD4+CD25highCD127lowCD45RA- (mTregs) and CD4+CD25highCD127lowCD45RA+ (nTregs) were analysed for FoxP3 expression. In suppression assays, nTregs and mTregs were cocultured with T responder cells (CD4+CD25-CD127+) at ratios of 1:1, 1:4 and 1:10 for 18 h. Proliferation was evaluated by using a BrdU assay.

Results:
Total Tregs were reduced in patients compared with controls, 3.8 (3.2-4.7) % vs 4.8 (4.0-5.4) %), p=0.011. Patients also had lower proportions of nTregs, 1.0 (1.0-1.3) % vs 1.4 (1.1-2.0) %, p=0.015, compared with controls while the difference in mTregs did not reach significance, 2.6 (2.0-3.3) % vs 3.0 (2.3-3.2) %. The expression of FoxP3+ in nTregs tended to be reduced in patients compared with controls (77 % vs 88 %, p=0.06) while its expression in mTregs was significantly decreased in patients compared with controls (83 % vs 90 %, p=0.015). The expression of CTLA4 and Helios was significantly higher in mTregs than nTregs in vivo but did not differ between patients and controls. However, the ability of Tregs, in particular nTregs, to inhibit responder CD4+CD25- T cell proliferation at a 1:1 ratio was significantly reduced in patients, p<0.001.

Conclusion:
The proportions of Tregs as well as FoxP3 expression in Tregs were reduced in CAD patients. Moreover, the suppressive capacity of nTregs was markedly impaired in patients although the expression of activation markers on Tregs in vivo did not seem to reflect the suppressive capacity ex vivo. The data highlight the presence of immune perturbations in CAD and call for further studies to evaluate clinical consequences involving immune dysfunction.
Abstract 347: Early Drug-Induced Inhibition of Proatherogenic Genes in Coronary Regions of Low Endothelial Shear Stress in Diabetic Hyperlipidemic Juvenile Swine
Authors: Papafaklis Michail I , Koskinas Konstantinos C , Sukhova Galina K , Baker Aaron B , Antoniadis Antonios P , Coskun Ahmet U , Franses Joseph W , Takahashi Saeko , Edelman Elazer R , Stone Peter H , Feldman Charles L
Published: March 2022

Introduction:
Low endothelial shear stress (ESS) activates pro-inflammatory pathways and is a powerful instigator of atherogenesis. Angiotensin receptor blockers and statins have been associated with anti-inflammatory actions in advanced plaques. However, their effect on the earliest pathobiologic manifestations of atherosclerosis has not been studied. We tested the hypothesis that valsartan (V) or V plus simvastatin (V/S) exerts an early vasculoprotective effect in coronary regions exposed to low ESS in a porcine model of human-like atherosclerosis.

Methods:
Twelve diabetic-hyperlipidemic swine (age: 3 mo) were grouped into controls (n=4), and those treated with V (320 mg; n=4) or V/S (320/40 mg; n=4). 3D reconstruction of coronary arteries by angiography and intravascular ultrasound was performed
in vivo
at 4 (baseline) and 8 (follow-up) wks post-induction. Baseline local ESS was calculated by computational fluid dynamics and 3 mm segments with low (≤1.2 Pa; n=46) or higher (>1.2 Pa; n=66) ESS were identified. Coronary arteries were harvested at follow-up. qRT-PCR was used for assessing the expression of intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), LDL receptor and lipoprotein-associated phospholipase-A
2
(LpPLA
2
).

Results:
The upregulation of ICAM-1, MCP-1, LDL receptor (p<0.05) and LpPLA
2
(p<0.1) expression in low ESS segments was inhibited in the V and V/S groups compared to controls (Figure).

Conclusion:
V and V/S attenuate the proatherogenic effects of low ESS within only 8 wks. These results suggest a drug-induced mechanism of regional atheroprotection early in the natural history of coronary artery disease.
Abstract 356: C/EBP α Mediates Shear Stress-Dependent Downregulation of the Angiotensin Type 1 Receptor
Authors: Simon David , Rivas Daniel , Lehoux Stephanie
Published: March 2022

The renin-angiotensin system, through the actions of angiotensin II, has significant proinflammatory actions on the vessel wall that can contribute to the progression of atherosclerosis. Hemodynamic forces are major determinants of atherosclerotic plaque localization. Plaques tend to form at arterial bifurcations, branch points, and curvatures where blood flow is low and oscillatory, whereas blood vessels exposed to high laminar shear stress remain comparatively devoid of plaques. Immunostaining of C57BL/6 mouse aortic arches showed a distinctive endothelial angiotensin type 1 receptor (AT1R) staining at the athero-prone inner curvature of the aortic arch but not in the adjoining outer curvature. In HUVECs exposed to laminar shear stress, we found that the AT1R was downregulated within 3 hours. The objective of the present study was to determine the mechanisms underlying shear stress-dependent downregulation of AT1R. We designed luciferase plasmids driven by different constructs of the AT1R promoter, spanning -1934 to +101 from the transcription start site, and transfected them into mouse primary endothelial cells. Using luciferase reporter assay, we found that shear stress reduced expression of the reporter construct by 42% (static = 3.00±0.51%, shear = 1.73±0.30%, P<0.05) after 6 hours. Using truncated constructs of the AT1R promoter, we identified the region -492 to -657 as necessary for the shear stress-dependent downregulation of AT1R (static = 1.20±0.23%, shear = 1.11±0.22%). Based on sequence conservation among human, mouse, rat and chimp, transcription factor binding site analysis pointed to a well-conserved site for C/EBP alpha. We used chromatin immunoprecipitation followed by qPCR analysis to demonstrate that C/EBP alpha was more strongly bound to the AT1R promoter under shear stress (3.21±0.83%) compared with static conditions (0.99±0.29%, P<0.05). Immunofluorescence also showed that C/EBP alpha was more abundant in the nucleus of endothelial cells under shear stress (12.7±3.0%) than static (1.9±1.6%) conditions. Our results suggest that the shear-stress dependent downregulation of AT1R is, at least in part, mediated through C/EBP alpha. This is, to our knowledge, the first indication of a role for C/EBP alpha in shear stress-dependent gene regulation.
Abstract 362: Akt-Dependent Interaction with EBP50 Regulates Skp2 Localization and Stability
Authors: Song Gyun Jee , Barrick Stacey , Leslie Kristen L , Bisello Alessandro
Published: March 2022

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout mice. We previously reported that the mitogenic effect of EBP50 is Akt-dependent and mediated by the S-phase kinase-associated protein 2 (Skp2), a component of the E3 ligase complex that degrades the cell cycle inhibitor p21cip1. The mechanisms by which Akt directly increases Skp2 stability are controversial. We hypothesize that EBP50 regulates Akt-dependent stabilization of Skp2. The C-terminal four amino acids of Skp2 (PSCL) are a canonical PDZ-binding sequence. Co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and Skp2. Mutation of the C-terminal Leu to Ala (L424A-Skp2) abrogated the interaction with EBP50. Akt potentiated the association between EBP50 and Skp2. Sequence analysis indicated the presence of an Akt phosphorylation motif (RPRLCT) in EBP50 between residues 151 and 156. Indeed, in vitro assays using purified EBP50 and constitutively active Akt showed that Akt phosphorylates EBP50. This effect was abrogated by a naturally occurring mutation R153Q in EBP50 that disrupts the Akt consensus sequence. EBP50 was expressed exclusively in the cytoplasm of VSMC, where it associated with the cytoskeleton. Cell fractionation and fluorescence microscopy studies showed that expression of EBP50, but not of R153Q, induced relocalization of Skp2 from the nucleus to the cytoplasm. EBP50 expression increased Skp2 stability and, consequently, VSMC proliferation. Collectively, these experiments show that Akt-mediated EBP50 phosphorylation increases the interaction with Skp2, regulates Skp2 cellular localization and increases Skp2 stability. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide the mechanistic basis for the effect of this scaffolding protein on VSMC proliferation and neointima formation.
Abstract 372: Regulation of Nox1 and Nox2 by Cigarette Smoke in Cerebral Vascular Smooth Muscle Cells: A Role of Oxidative Stress in Cerebral Aneurysm Formation
Authors: Ali Muhammad S , Jabbour Pascal M , Tjoumakaris Stavropoula I , Gonzalez L Fernando , Rosenwasser Robert H , Koch Walter J , Dumont Aaron S
Published: March 2022

Objectives:
Cigarette smoke is one of the most significant environmental risk factor for cerebral aneurysm formation and progression. Factors including inflammation and matrix degradation are considered to be important in aneurysm formation. Reactive Oxygen Species (ROS) play a pathophysiological role in vascular inflammation, proliferation, migration and angiogenesis. Among the major sources of ROS in vascular smooth muscle cells (VSMCs) are NAD(P)H oxidase (Nox). We investigated the role of Cigarette Smoke Extract (CSE) in regulation of Nox1 and Nox2 isoforms in cerebral VSMCs both in vitro and in vivo.

Methods:
Rat cerebral VSMCs were treated with CSE at 10 and 40 ug/ml for 4 and 24 hours. The level of water soluble components of cigarette smoke and nicotine levels present in the CSE overlap with plasma levels of these constituents in human smokers. RNA was extracted and qPCR was performed. Cerebral VSMCs were transfected with siNox1 and siNox2 for 24 and 72 hours and further incubated with CSE for 24 hours. As part of in vivo studies, Pluronic Gel (40% w/v) containing 0.2-0.8 mg/ml of CSE was applied to the adventitial surface of rat carotid arteries for 24 hours. Vessels were harvested for mRNA quantification.

Results:
CSE decreased the expression of VSMC marker genes and myocardin, increased expression of KLF4 (a transcription factor) and proinflammatory/matrix remodeling marker genes including MMPs, VCAM1 and MCP1. In addition CSE increased the expression of both Nox1 and Nox2 at 4 and 24 hours but was more significant at 4h. Preliminary studies with siNox1 and siNox2 decreased the expression of KLF4, and reversed inflammatory markers gene expression caused by CSE stimulation. siNox1 increased the expression of marker genes including alpha-Actin and 22-alpha. Expression of Nox1 and Nox2 was markedly increased following in vivo application of pluronic gel.

Conclusions:
These findings indicate that cerebral VSMCs contain Nox1 and Nox2, are potently regulated by CSE and play an important role in NAD(P)H oxidase driven ROS production. CSE induces marked phenotypic modulation of vertebral VSMC while concomitantly increasing expression of Nox1 and Nox2. Furthermore, alteration of Nox expression abrogates aspects of CSE induced VSMC phenotypic modulation. These molecular changes implicate oxidative stress in vascular pathology underlying cerebral aneurysms and may provide a potential target for future therapeutic strategies.
Abstract 38: Segment-Specific Association of Body Mass Index with Carotid Plaque and Intima-Media Thickness: The Northern Manhattan Study
Authors: Dong Chuanhui , Cabral Digna , Marquez Chensy , Elkind Mitchell S , Sacco Ralph L , Rundek Tatjana
Published: March 2022

Objective:
Obesity is a risk factor for cardiovascular disease and stroke, and carotid intima-media thickness (IMT) and plaque are markers of subclinical atherosclerotic disease. The differential relationship of body mass index (BMI) to IMT and plaque at different carotid segments has not been studied. In the present analysis, we evaluated the segment-specific association of BMI with IMT and plaque presence in a community-based cohort from the Northern Manhattan Study (NOMAS).

Methods and Results:
A total of 1653 NOMAS subjects (mean age 69±9 years, 40% male, 18% white, 20% black and 62% Hispanic) underwent B-mode ultrasound examination of the common carotid artery (CCA), internal carotid artery (ICA) and bifurcation (BIF). Generalized linear modeling was performed to evaluate the relations of BMI to IMT or plaque presence in CCA, ICA and BIF, adjusted for age, sex, race-ethnicity, smoking, physical activity, alcohol drinking, systolic and diastolic blood pressure, fasting glucose, LDL, HDL, medications for hypertension, diabetes, and high cholesterol. Overall, the mean BMI was 28±5 kg/m2 (29% obese, 45% overweight). The mean IMT was 0.73±0.09 mm in CCA, 0.64±0.08 mm in ICA, and 0.74±0.10 mm in BIF. The prevalence of plaque was 9% in CCA, 20% in ICA and 53% in BIF. The change in IMT (mm) for per unit BMI increase was largest in CCA (Beta=0.002, P<0.0001), intermediate in ICA (Beta=0.001, P=0.002), and smallest in BIF (Beta=0.0006, P=0.22). Similarly, compared with those with normal weight, obese individuals had larger IMT in CCA (Beta=0.03, P<0.0001) and ICA (Beta=0.02, P=0.01) but not in BIF (Beta=0.006, P=0.40). No association was found between BMI and plaque presence in CCA (P=0.74), ICA (P=0.69), or BIF (P=0.94). There was no significant effect modification by race-ethnicity or sex on the association of BMI with IMT or plaque presence at a specific segment.

Conclusion:
BMI may be a risk factor for IMT in CCA and ICA rather than BIF or presence of plaque at any specific segment. Given that these carotid segments differ in geometry, shear stress, extracellular matrix structure, and cell composition, further studies are needed to explore the differences in mechanisms underlying the development of atherosclerosis lesions in different carotid segments.
Abstract 390: Cardiovascular Risk Factors and Endothelial Dysfunction Cause Rarefaction of the Collateral Circulation and Impaired Recovery from Ischemia
Authors: Moore Scott M , Waters Matthew R , McFadden William A , Faber James E
Published: March 2022

Aging and cardiovascular risk factors (CVRF) such as diabetes, hypertension, dyslipidemia and metabolic syndrome have been associated with collateral insufficiency in animals and humans. Long-standing CVRF as well as collateral insufficiency associate with poorer outcomes following acute ischemic stroke or myocardial infarction. Evidence suggests that endothelial dysfunction (decreased eNOS activity and NO bioavailability) is caused by and contributes to CVRF, and promotes atherosclerosis and thrombosis in patients with CVRF. Aging and eNOS deficiency have been shown to cause rarefaction of native (pre-existing) cerebral and hindlimb collaterals in mice and to reduce collateral remodeling in response to arterial occlusion. To determine whether endothelial dysfunction and CVRF cause collateral rarefaction and/or impair remodeling, we examined recovery of perfusion following femoral artery ligation (FAL) and native cerebral pial collateral extent (density and diameter) in the following mouse models (all 7-8 mos-age): chronic L-NAME treatment (endothelial dysfunction),
ob/ob
(obesity), LDLR
-/-
on high-fat diet (hyperlipidemia), LDLR
-/-
;
ob/ob
(metabolic syndrome), RenTgMK
+/-
(hypertension), and
Akita
(type-1 diabetes). Hindlimb perfusion immediately after FAL (dependent on native collateral extent) and recovery over 21 days (dependent on collateral remodeling) were impaired by chronic endothelial dysfunction, hypertension, metabolic syndrome, and diabetes (p<0.05) but not by obesity and hyperlipidemia. Similarly, native pial collateral extent was significantly reduced in models of endothelial dysfunction, hypertension, metabolic syndrome and diabetes. The functional significance of CVRF-induced collateral rarefaction was evident as more severe ischemic injury and use impairment in hindlimb and larger infarction in brain after middle cerebral artery occlusion. Investigations into the mechanisms of CVRF-induced collateral rarefaction and treatments to prevent it are ongoing. These data show that CVRF not only impair collateral remodeling but also cause significant loss of native collaterals, resulting in worse ischemic tissue injury after acute obstruction and fewer collaterals available for remodeling.
Abstract 4: Apolipoprotein (apo) A-I Increases Insulin-Dependent Glucose Disposal in Skeletal Muscle by Stimulating the PI3K/Akt/AS160 Signaling Pathway
Authors: Tang Shudi , Barter Philip , Rye Kerry-Anne
Published: March 2022

Introduction:
Inhibition of CETP increases plasma high density lipoprotein (HDL) levels and improves glycaemic control in people with type 2 diabetes. Recent in vitro studies have also established that the main HDL apolipoprotein, apoA-I, increases insulin secretion from pancreatic β-cells. However, the effect of apoA-I on glucose uptake by skeletal muscle, the main site of glucose disposal, is not understood.

Hypothesis:
Lipid-free apoA-I increases insulin-dependent glucose disposal in skeletal muscle.

Methods:
Primary human skeletal muscle cells were differentiated into myotubes, then incubated for 16 h with lipid-free apoA-I (final concentration 1 mg/ml). Glucose uptake was initiated by adding a tracer amount of [3H]2-deoxy-glucose to the myotubes and continuing the incubation for up to 5 h with or without insulin (0.1 μM final conc.). IRS-1, PI3K, Akt and AS160 protein levels were quantified by subjecting total cell lysates to SDS-PAGE and immunoblotting.

Results:
Incubation of skeletal muscle myotubes in the absence or presence of insulin resulted in [3H]2-deoxy-glucose uptake of 1000 and 1103±68.9 pmol/mg protein/h, respectively (p<0.1). Pre-incubation of the myotubes with lipid-free apoA-I, followed by incubation in the absence or presence of insulin resulted in [3H]2-deoxy-glucose uptake of 1089±51.35 and 1358±57.9 pmol/mg protein/h (p<0.01), respectively. The apoA-I-mediated increase in insulin-dependent glucose uptake into the myotubes was time- and concentration-dependent. Incubation of myotubes in the presence of apoA-I and insulin increased IRS-1 phosphorylation and total IRS-1 protein by 27.8±5% and 30.8±1.3% (p<0.01 for both), respectively. PI3K, Akt and AS160 phosphorylation increased by 55.1±22%, 52.5±20.9%, and 32±8%, respectively (p<0.01 for all) without any change in total protein.

Conclusion:
Lipid-free apoA-I increases insulin-dependent glucose uptake into human skeletal muscle cells by stimulating the PI3K/Akt/AS160 insulin signaling pathway. These results suggest that HDL-raising therapies may improve glucose disposal in skeletal muscle, and thus be beneficial in type 2 diabetes.
Abstract 429: High-Risk Features of Carotid Atherosclerotic Disease Occur Bilaterally: A Cross-Sectional, High-Resolution MRI Investigation
Authors: Yamada Kiyofumi , Hippe Daniel S , Underhill Hunter R , Ferguson Marina S , Sun Jie , Chu Baocheng , Yuan Chun , Hatsukami Thomas S
Published: March 2022

Background:
The presence of intraplaque hemorrhage (IPH), large lipid rich necrotic core (LRNC), and fibrous cap rupture (FCR) in the carotid plaque as detected by magnetic resonance imaging (MRI) has been associated with future cerebrovascular events. This study sought to determine whether these high-risk features occur symmetrically in the carotid arteries.

Methods:
Multi-contrast MR images of both carotids from 119 patients with 50- 79 % stenosis in at least one carotid artery were reviewed. Images from each artery were interpreted independent from results of the contralateral artery. Images were reviewed for presence/absence of LRNC, IPH and FCR. The maximum size of LRNC relative to the wall (%LRNC) was also recorded.

Results:
Table 1 shows the significantly increased probability of having IPH and LRNC on the contralateral side when present on one side while FCR was nearly significant. In patients with LRNC present, there was a strong correlation in size of %LRNC between sides (Pearson’s r = 0.60, p<0.001). However, there was a significant difference in mean size of %LRNC between the two sides (30.0 vs. 12.8 %, p<0.001; paired t-test).

Conclusion:
These results provide compelling evidence that high-risk features of atherosclerotic disease are prone to develop bilaterally, although not concurrently, in patients with carotid disease. Presence of unstable features in one artery appears to herald the development of similar features in the contralateral artery which suggests that the pathophysiology of the high risk plaque is governed by systemic factors.
Abstract 43: The Metalloprotease ADAMTS13 Reduces VWF-Mediated Vascular Inflammation and Early Development of Atherosclerosis in Mice
Authors: Chauhan Anil K , Gandhi Chintan , Lentz Steven R
Published: March 2022

Background and objective:
ADAMTS13 prevents spontaneous thrombosis in the microvasculature by cleaving hyperactive ultra large von Willebrand factor (ULVWF) multimers into smaller and less active forms. Reduced ADAMTS13 activity and increased VWF levels have been described in many diseases associated with systemic inflammation. We hypothesized that, by cleaving ULVWF multimers and/or VWF multimers, ADAMTS13 reduces vascular inflammation and the development of early atherosclerotic plaques.

Model and methods:
ApoE
-/-,
Adamts13-/-/ApoE-/-, Adamts13
-/-/
Vwf
-/-/
ApoE
-/-
and
Vwf
-/-/ApoE
-/-
mice were fed a high-fat Western diet (20% fat, 0.2% cholesterol) beginning at 6 weeks of age until they were sacrificed at 4 months. Intravital fluorescence microscopy was used to visualize leukocyte adhesion and plaque formation at the carotid sinus. We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and
en face
lesion area measured by morphometry, and in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain. Inflammatory cells in the aortic lesions were quantitated by immunohistochemistry.

Results:
Using intravital microscopy, we observed a 4-fold increase (
P
<0.001) in leukocyte adhesion and at the carotid sinus of
Adamts13-/-/ApoE
-/-
mice compared with
ApoE
-/-
mice. Interestingly, 100% of the
Adamts13-/-/ApoE
-/-
mice had larger plaque (occluded vessel by ∼70-80 %) at the carotid sinus whereas only 20% of the
ApoE-
/- mice had plaques that were smaller in size (
P
<0.0001). Next, we found that the atherosclerotic lesions in the aorta and aortic sinus of the
Adamts13-/-/ApoE-/-
mice were not only larger (2-fold,
P
<0.001) but also more inflammatory (increased neutrophil and macrophage infiltration) with decreased interstitial collagen compared with
ApoE
-/-
mice, suggesting that ADAMTS13 reduces vascular inflammation and subsequent atherosclerotic plaque formation.
Adamts13-/-/ApoE
-/-
mice fed a normal chow diet also demonstrated significantly accelerated atherosclerotic plaque formation compared with
ApoE
-/-
mice. Total cholesterol and triglyceride levels were similar among groups fed high-fat Western or normal chow diets. Finally, atherosclerotic lesions in
Adamts13-/-/Vwf-/-/ApoE
-/-
mice were similar to
Vwf-/-/ApoE
-/-
mice, demonstrating that the accelerated atherosclerosis observed in ADAMTS13-deficient mice is VWF-dependent.

Conclusion:
These findings reveal a new role for the antithrombotic enzyme ADAMTS13 in reducing VWF-mediated plaque formation during early atherosclerosis.
Abstract 462: Dose Matters: Omega-3 Polyunsaturated Fatty Acid Supplementation at 3600 mg/d but Not 900 mg/d Reduces Inflammatory Response to Evoked Endotoxemia in Healthy Volunteers
Authors: Ferguson Jane F , Mulvey Claire K , Shah Rhia , Patel Parth , Kong Stephanie , Tabita-Martinez Jennifer , Terembula Karen , Mehta Nehal N , Shah Rachana , Reilly Muredach P
Published: March 2022

Inflammation is a key component in the pathogenesis of cardiometabolic disease. Certain anti-inflammatory compounds may improve atherosclerotic disease risk by modulating systemic and vascular inflammation. Long-chain omega-3 polyunsaturated fatty acids (n-3 PUFA) may represent a dietary mechanism for improving cardiometabolic health through reduction of inflammation. We hypothesized that n-3 PUFA supplementation would reduce the inflammatory response to evoked endotoxemia in healthy humans.

In the Fenofibrate and omega-3 Fatty Acid Modulation of Endotoxemia study (FFAME Study), we recruited healthy individuals (N=60; age 18-45; BMI 18-30; 43% Female; 65% Caucasian, 20% African, 15% Asian ancestry) randomized to one of three treatment groups; n-3 PUFA (Lovaza 465mg EPA/375mg DHA) supplemented at either 900mg per day or 3,600mg per day, or placebo. An additional arm of the study included a fenofibrate treatment group, not included here. Participants were treated for ∼8 weeks then admitted to the research center for an endotoxin challenge of lipopolysaccharide (LPS) 0.6ng/kg intravenously. Multiple clinical and biochemical variables were assessed throughout, and analyzed using RM-ANOVA with Bonferroni post-hoc tests for between group differences.

Compared to placebo, high-dose (P<0.01) but not low dose n-3 PUFA reduced peak temperature, but not heart rate or blood pressure, responses to endotoxin. Cytokines and inflammatory markers (IL-6, TNFα, IL-10, CRP, SAA) were decreased in high-dose n-3 PUFA vs. placebo . Interestingly, these inflammatory markers tended to be increased in low-dose n-3 PUFA vs. placebo, with a significant difference in the effect of low-dose vs. high-dose n-3 PUFA on peak IL-6 (P<0.001), TNFα (P<0.01) and IL-10 (P<0.05). Plasma lipids tended to decrease in both n-3 PUFA groups compared with placebo, with equal lipid-lowering effect of 900 or 3,600 mg/day on cholesterol, ApoA-I, and triglycerides.

The specific anti-inflammatory properties of n-3 PUFA appear to be dose-specific, with high doses (3,600mg/day) leading to attenuation of the inflammatory response to evoked endotoxemia, while lower doses (900mg/day) do not reduce the inflammatory response despite similar reductions in plasma lipids.