bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 464: Progesterone Signaling in Endothelial Cells Results in Increased Vascular Permeability and Platelet Adhesion
Authors: Goddard Lauren M , Murphy Thomas J , Enciso Josephine M , Sanchez Lauren , Iruela-Arispe M Luisa
Published: July 2021

Epidemiological studies have indicated the rather significant, yet unclear, impact of sex hormones on cardiovascular physiology and disease. Both observational studies and clinical trials have produced conflicting evidence as to the benefits of female sex hormones, particularly with regards to progesterone. Prolonged exposure to progesterone leads to break-through bleeding, vessel fragility and venous thromboembolisms. Currently, little is known on the molecular and cellular effects of progesterone on the cardiovascular system, or its cell specific functions. In fact, the vascular effects attributed to progesterone have been viewed, for the most part, as secondary consequences of its multiple systemic activities. Using a PRLacZ reporter mouse we have determined that progesterone receptor (PR) expression in the vasculature is restricted to endothelial cells of veins and lymphatics of the reproductive tract. Conditional deletion of PR from endothelial cells using VE-cadherin Cre leads to decreased vascular permeability in the uterus and ovary following treatment with progesterone. Transgenic misexpression of PR in the lung results in pathological vascular leakage and intravascular platelet adhesion in response to progesterone. Additionally, activation of PR within endothelial cells leads to the development of stress fibers, formation of inter-cellular gaps, increased permeability and changes in expression of cell-cell junctional proteins. These results reveal a previously unknown and direct role for PR on endothelial cell function with consequences for physiological permeability, blood vessel integrity and homeostasis.
Abstract 466: Role of Janus Kinases and Signal Transducers and Activators of Transcription in Interferon-gamma--Induced Insulin Resistance in Human Adipocytes
Authors: O’Neill Sean M , Hinkle Christine C , Reilly Muredach P
Published: July 2021

Obesity induces chronic low-grade inflammation of the adipose tissue, accompanied by an infiltration of cytokine-secreting immune cells, driving adipose and systemic insulin resistance. Circulating levels of interferon-gamma (IFNγ) in obesity correlate with increased adiposity and insulin resistance. Furthermore, IFNγ inhibits insulin signaling in adipocytes. IFNγ receptors signal via the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway, but the specific molecules involved with IFNγ signaling in adipocytes have not been determined. Based on pharmacological data, we hypothesized that JAK1 and STAT1, but not JAK2 and STAT3 would mediate IFNγ signaling in human adipocytes. To test this hypothesis we developed lentivirus-delivered shRNA against JAK1, JAK2, STAT1, and STAT3. Knockdown of JAK1, JAK2, and STAT1 in adipocytes with shRNA significantly inhibited IFNγ-induced suppressor of cytokine signaling (SOCS)1 and SOCS3 expression. IFNγ downregulated insulin-signaling related gene (insulin receptor substrate-1, insulin receptor, and glucose transporter-4), and adipogenic gene (adiponectin, fatty acid synthase, lipoprotein lipase, perilipin, and peroxisome proliferator-activated receptor gamma) expression. JAK1, JAK2, and STAT1 shRNA attenuated IFNγ-induced downregulation of insulin signaling related and adipogenic gene expression, whereas STAT3 shRNA had no significant effect. These findings suggest that, in adipocytes, JAK1 and JAK2 as well as STAT1, but not STAT3, contribute to IFNγ signaling and the subsequent decrease in insulin sensitivity. Understanding the molecular mechanisms by which inflammation induces adipose insulin resistance may identify pathways for therapeutic treatment or prevention of obesity induced insulin resistance and diabetes.
Abstract 467: The ApoA-1 Mimetic Peptide D-4F Shows Antidiabetic Efficacy In db/db Mice Only After Intraperitoneal Administration
Authors: Rolin Bidda , Kirk Rikke K , Gotfredsen Carsten , Bjerring Barbara H , Klitgaard Thomas , Kodra Janos T , Mohr Malene , Rasmussen Salka E
Published: July 2021

Objective:
The apoA-I mimetic peptide L-4F has previously been shown to have antidiabetic efficacy in ob/ob mice. In this study we tested the antidiabetic potential of the D-4F peptide.

Methods:
We dosed 6 weeks old male db/db mice once daily with 4.5 μmol/kg D-4F either given by the ip. or the sc. route. Blood glucose (BG) and body weight (BW) were monitored twice weekly, 24 h food intake was measured daily and HbA1c was measured once weekly. After 5 weeks the mice were sacrificed and blood was collected for D-4F concentration measurements. Abdominal and subcutaneous tissues were harvested for histology.

Results:
Blood glucose, HbA1c and food intake were all significantly reduced in the group dosed with D-4F by the ip route as compared to the vehicle dosed group. In contrast, there was no effect of D-4F when given by the sc. route. This was despite similar and high plasma concentration of D-4F (Table 1). Macroscopially, chronic adhesive peritonitis was evident in the ip dosed group and histological evaluation of the peritoneal tissue covering abdominal fat confirmed this finding. In the group dosed sc, chronic inflammation was also observed at the injection site, both histologically and by macroscopic evaluation.

Conclusion:
Ip dosing of D-4F gave rise to chronic peritonitis, which could have caused the reduced food intake and consequently the reduction in blood glucose and HbA1c. This effect was not observed when D-4F was injected sc, despite the local tissue reaction in subcutis. Therefore, we speculate that the antidiabetic effect of D-4F observed after ip administration is an artefact due to abdominal inflammation and reduced food intake.

Table 1. Means±SEM

§
Vehicle ip from a separate study; * p<0.05, **p<0.01, ***p<0.001 (Two way ANOVA).
Abstract 468: The Role of Ca2+/PKA Signaling Enhancer Protein Phosphatase Inhibitor 1 in the Control of Ca2+ Cycling and Signaling in VSMCs
Authors: Hadri Lahouaria , Bobe Regis , Lopez Jose J , Turnbull Irene C , Kovacic Jason C , Karakikes Ioannis , Kranias Evangelia , Hajjar Roger J , Larissa Lipskaia
Published: July 2021

Vascular remodeling is associated with trans-differentiation of contractile vascular smooth muscle cells (VSMC) towards a proliferating/synthetic phenotype. We have recently demonstrated (Bobe et al., 2011) that the Ca2+ cycling in “contractile” VSMC requires the expression of the fast isoform of sarco/endo plasmic ATPase (SERCA2a), whereas the Ca2+ cycling in “proliferating” ones is associated with the expression of the ubiquitous isoform SERCA2b only. Phospholamban (PLB), a negative regulator of SERCA2 activity, is inhibited by PKA phosphorylation and activated by protein phosphatase 1 (PP1) dependent dephosphorylation. Inhibitor-1 (I-1), a highly specific inhibitor of PP1, enhances both PKA-dependent PLB phosphorylation and SERCA activity.

The goal of this study was to elucidate the role of PKA signaling enhancer I-1 in the control of VSMC Ca2+ cycling.

In humans and rodents, the expression of I-1 was found to be specific for contractile VSMC, whereas its target PP1 was highly expressed in synthetic VSMC. Consequently, PLB phosphorylation was decreased in synthetic VSMC whereas the expression of total PLB remained unchanged. Genetic deletion of I-1 in mice model (I-1 KO) resulted in lack of PLB phosphorylation in VSMC of adult animals. Despite the fact that SERCA2a was expressed, VSMCs from I-1 deficient mice were locked in the synthetic state. Consistent with this, the adult I-1 deficient mice developed a vascular proliferative disorder and excessive neointimal proliferation after vascular injury. Adenovirus-directed gene transfer of constitutively active I-1 (I-1c) significantly increased PLB phosphorylation and Ca2+ uptake in both synthetic and contractile VSMCs, however, there was no effect on the type of Ca2+ transient, which appears to be a SERCA2 isoform-dependent characteristic. Therefore, I-1c prevented proliferation and remodeling of contractile VSMC, but had no effect on synthetic VSMC, which predominantly express SERCA2b.

In conclusion, synergistic action of I-1 and SERCA2a is necessary for acquisition of the VSMC contractile phenotype. Gene transfer of I-1c may be considered as a promising therapeutic strategy for preventing vascular proliferative diseases.
Abstract 474: Effects of Cigarette Smoke on CD146 Expression and Function in Pulmonary Endothelial Cells
Authors: Kratzer Adelheid , Salys Jonas , Gonzalez Benjy , Chu Hong Wei , Zamora Martin , Taraseviciene-Stewart Laima
Published: July 2021

Background and Objectives:
Cell adhesion molecule CD146 is a transmembrane glycoprotein constitutively expressed in all types of endothelial cells (EC). It exists in two forms: a membrane-anchored form (CD146) and a soluble, extracellular and cleaved form (sCD146). The plasma concentration of sCD146 is modulated in inflammatory diseases that involve endothelial alterations. We investigated the role of endothelial CD146 in cigarette smoke-induced emphysema
in vivo
and in pulmonary endothelial cells (EC)
in vitro
.

Methods:
Sprague Dawley rats exposed to cigarette smoke for 2 months developed significant emphysematous changes (measured by mean linear intercept). Levels of sCD146 were subsequently measured in the circulation as well as in the bronchoalveolar lavage fluid (BALf) via ELISA.
In vitro
studies were carried out in rat pulmonary microvascular endothelial cells using CSE.

Results:
CD146 is highly expressed in rat pulmonary microvascular endothelial cells (RPMVEC) and to a much lower extent, in pulmonary macrovascular endothelial cells (RPAEC). Treatment of RPMVEC with cigarette smoke extract (CSE)
in vitro
resulted in decreased expression of membrane-bound CD146 as well as a reduced gene expression and increased sCD146 levels in the culture medium after 12 hours. Moreover, CSE-induced downregulation of CD146 expression resulted in increased vascular permeability of RPMVEC, as measured by EVANs Blue assay and migration of CFSE-labeled rat alveolar macrophage. Immunofluorescent staining revealed that CSE treatment resulted in translocation of membrane-bound CD146 into the nucleus. Subsequent western blot analysis showed changes in ERK and AKT activation and signaling. Similar results were found upon siRNA silencing of CD146, implicating a role for CD146 in tissue inflammation and integrity. Circulating levels of sCD146 were also elevated in plasma and BALf of patients with COPD and correlated, in part, with the presence of anti-endothelial autoantibodies. Additionally, we found decreased expression of membrane-bound CD146 in lung tissues of COPD patients.

Conclusions:
Our data suggest that CD146 plays an important role in pulmonary vascular EC function. Moreover, levels of circulating soluble CD146 can be a predictor of vascular endothelial cell injury.
Abstract 477: SM22 Deficiency Induces SRF Nuclear Translocation and Promotes Carotid Medial Fibrosis in Response to Injury
Authors: Shen Jianbin , Ju Donghong , Xie Youming , Jiang Hong , Li Li
Published: July 2021

Introduction:
SM22 (aka transgelin) is known to be an actin-binding protein that is rapidly downregulated in response to vascular injury. Despite prevalent view regarding SM22 as a maker of VSMC dedifferentiation, recently a series of studies including our own demonstrate that SM22 downregulation promotes SMC dedifferentiate into a variety of SMC subtypes involving in proliferation, migration, inflammation, osteochondrogendesis and apoptosis.

Hypothesis:
In the present study, we assess the hypothesis that SM22 regulates the expression of col1a2, a fibrotic marker, in SMCs.

Methods:
We use molecular and cellular approaches to analyze the regulation of col1a2 in smooth muscle cells upon artery injury using Sm22 knockout mice.

Results:
We found that carotid injury induces high expression of Col1a2 in the medial layer of the vessel wall and the nuclear translocation of serum response factor (SRF), a key transcription factor that regulates muscle-specific genes as well as growth factor response genes. To determine whether
col1a2,
an extracellular matrix protein, is a target of SRF, we performed bioinformatics analysis: we identified an evolutionarily conserved SRF binding sequence, CC(A/T)6GG (known as the CArG box), in the col1a2 promoter. Luciferase transfection assay showed that Vp16-SRF significantly transactivated the col1a2 promoter; mutation of the CArG box abolished most of this stimulating effect. Gel shift and chromatin precipitation assays confirmed that SRF bound to the CArG box of the
col1a2
promoter. To investigate whether loss of SM22 in SMCs contributes to increased nuclear SRF expression, we knocked down SM22 in PAC1 cells, a VSMC cell line, using SM22 siRNA. Consistent with the in vivo observation, knockdown SM22 also enhanced nuclear SRF expression. However,
cola2
transcription was not upregulated, suggesting that additional signals may be involved in this regulation
in vivo
.

Conclusions:
SM22 deficiency increases the fibrosis in the arterial media, and the induction of nuclear SRF may contribute at least in part to
col1a2
transcription in response to artery injury.
Abstract 482: Peroxisome Proliferator-Activated Receptor α Modulation with Fenofibrate Does Not Alter the Clinical or Inflammatory Responses to Evoked Endotoxemia in Humans
Authors: Mulvey Claire K , Ferguson Jane F , Terembula Karen , Tabita-Martinez Jennifer , Shah Rhia , Patel Parth , Usman M H , Shah Rachana , Mehta Nehal N , Rader Daniel J , Reilly Muredach P
Published: July 2021

Introduction:
Inflammation plays a crucial role in the pathophysiology of cardiometabolic disease. Some experimental data and clinical trials suggest that PPAR-α activation and fibrate therapy suppress inflammation in humans, potentially promoting anti-atherogenic actions. Human experimental endotoxemia provides a controlled model for the study of atherogenic inflammatory responses and their modulation
in vivo
. We hypothesized that fenofibrate would suppress the inflammatory responses to endotoxin (LPS) in healthy humans.

Methods:
In the Fenofibrate and Omega-3 Fatty Acid Modulation of Endotoxemia study, 36 healthy volunteers (mean age 26 ± 7 years; mean BMI 24 ± 3 kg/m
2
; 56% male; 72% Caucasian, 19% African, 8% Asian ancestry) were randomized to fenofibrate 145 mg or placebo daily. After 8 weeks of treatment, subjects underwent a low-dose LPS challenge (0.6 ng/kg intravenous bolus). Anthropometric, clinical, and blood measures were collected at baseline, pre-LPS, and serially for 24 hours after LPS exposure.

Results:
Compared to placebo, fenofibrate reduced total cholesterol (170 ± 35 to 137 ± 31 mg/dL, p=0.009) and tended to decrease triglycerides (78 ± 40 to 57 ± 23 mg/dL, p=0.611) prior to LPS. LPS induced a modest clinical response with increased temperature, heart rate, and blood pressure (
p
<0.001 for all). Cytokines and chemokines increased significantly, peaking 2 to 4 hours post-LPS (TNFα 12-fold, IL-6 13-fold, IL-10 9-fold, MCP-1 10-fold;
p
<0.001 for all). Acute phase reactants also increased, peaking at 24 hours (serum amyloid A (SAA) 14-fold, C-reactive protein (CRP) 9-fold; both
p
<0.001). Compared to placebo, fenofibrate had no effect on LPS-induced clinical responses, cytokine or chemokine release as measured by peak response or area under the curve (AUC). Fenofibrate also had no effect on plasma CRP or lipid parameters following LPS. Although peak SAA and SAA AUC tended to be lower with fenofibrate vs. placebo, the differences were not statistically significant (
p
=0.08 for both).

Conclusions:
PPAR-α modulation with fenofibrate did not attenuate clinical or inflammatory responses during low-grade human endotoxemia. These data suggest the anti-inflammatory properties of fenofibrate at clinically-relevant dosing are limited.
Abstract 483: Shear Stress-Induced Proinflammatory Responses on Transitional Matrices Require αv Integrins but Not α5β1
Authors: Chen Jie , Mazar Andrew P , Orr Wayne
Published: July 2021

The frictional shear stress generated by blood flow activates integrins on the endothelial cell surface, and signaling downstream of newly activated integrins regulates several flow-induced responses including inflammation and permeability. The extracellular matrix ligands for these integrins are altered early during atherosclerosis, as transitional matrix proteins such as fibronectin and fibrinogen accumulate in the subendothelial matrix. Signaling through transitional matrix binding integrins (α5β1, αvβ3 and αvβ5) drives shear stress-induced NF-κB activation and proinflammatory gene expression (ICAM-1, VCAM-1); however the specific contribution of each of these integrins remains unclear. We now show that α5 and αv expression occurs concomitant with early endothelial cell activation during atherosclerotic plaque formation in both mice and humans. Preventing αv integrin signaling (small molecule inhibitor S247, siRNA) abolished shear stress-induced proinflammatory signaling (p21-activated kinase (PAK), NF-κB), whereas preventing α5 integrin signaling (ATN-161, siRNA) did not. Furthermore, αv knockdown inhibited flow-induced ICAM-1 and VCAM-1 expression (both mRNA and protein) following either short term (5 hr) laminar and long term oscillatory (24 hr) shear stress. In contrast, αv knockdown did not affect TNFα-induced ICAM-1 and VCAM-1 expression, suggesting the anti-inflammatory effect of αv inhibition is specific to shear stress. Integrin inhibition did not affect all shear stress responses, as neither α5 nor αv siRNA affected shear stress-induced extracellular-signal regulated kinase (ERK1/2) activation. Taken together, these studies suggest that αv integrins (αvβ3, αvβ5) mediate shear stress-induced proinflammatory responses associated with the transitional matrix.
Abstract 484: Acidic pH Triggers Secretion of Proinflammatory Cytokines in Human Macrophages
Authors: Rajamäki Kristiina , Nordström Tommy , öörni Katariina , Åkerman Karl E , Kovanen Petri T , Eklund Kari K
Published: July 2021

Extracellular acidification can occur in atherosclerotic lesions, specifically in areas with high numbers of metabolically active macrophages secreting lactate and hydrogen ions. There is a growing body of evidence showing that acidification of the extracellular environment causes fundamental changes in the functions of immune cells.

Here we studied the effect of low pH on the secretion of proinflammatory cytokines by cultured human macrophages. Using custom manufactured cell culture media with neutral and acidic pH (pH 7.5-6.0) we discovered that acidic pH triggers secretion of interleukin(IL)-1beta and IL-18, but not of IL-1alpha. IL-1beta and IL-18 are both produced as proforms that are processed into the biologically active secreted form by the caspase-1 protease. The IL-1beta secretion induced by acidic pH was paralleled by caspase-1 activation, and inhibition of caspase-1 completely abolished IL-1beta secretion. Acidic pH-induced IL-1beta secretion was also abolished at high extracellular potassium concentrations, which implies that potassium efflux from cells is a crucial step upstream of caspase-1 activation. We are currently exploring the involvement of inflammasomes in the acidic pH -induced caspase-1 activation.

In conclusion, acidic microenvironments of atherosclerotic lesions may induce proinflammatory cytokine secretion in macrophages, so amplifying the progression of atherogenesis.
Abstract 487: EBP50 Promotes Epidermal Growth Factor-Dependent Activation of Focal Adhesion Kinase and Vascular Smooth Muscle Cells Migration
Authors: Song Gyun Jee , Leslie Kristen L , Barrick Stacey , Bougoin Sylvain , Bisello Alessandro
Published: July 2021

The Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) is a scaffolding protein that regulates a variety of physiological functions. Previous studies showed that EBP50 promotes vascular smooth muscle cells (VSMC) proliferation and neointima formation following arterial injury. In this study the role of EBP50 on VSMC migration was characterized. VSMC migration was determined by wound-healing assays. The motility of primary VSMC isolated from EBP50 KO mice was significantly reduced compared to WT cells (25 um/h WT vs. 19 um/h KO, p<0.05) resulting in a 50% reduction in wound coverage. Conversely, EBP50 expression increased VSMC migration. EBP50-null VSMC had fewer and larger focal adhesions than EBP50-expressing cells. Both assembly and disassembly of focal adhesion in response to epidermal growth factor (EGF) (determined by real time TIRF microscopy) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with both focal adhesion kinase (FAK) and EGF receptor (EGFR) and the formation of a complex containing both EGFR and FAK was increased by EBP50. Stimulation of VSMC with EGF induced FAK phosphorylation on Tyr925 in WT, but not in KO cell, whereas phosphorylation of Tyr397 was unaffected. Collectively, these observations indicate that EBP50 facilitates growth factors-dependent activation of FAK, and consequently, migration of VSMC. Therefore, in addition to increasing VSMC proliferation, EBP50 promotes neointima formation following arterial injury by increasing VSMC motility.
Abstract 490: Distribution of Oxidized Phospholipids and Lipoprotein-Associated Phospholipase A2 in Plasma, Density Gradient Fractions and Individual Lipoproteins in Patients with Familial Hypercholesterolemia: Effect of LDL Apheresis
Authors: Arai Kiyohito , Orsoni Alexina , Miller Elizabeth R , Mallat Ziad , Tedgui Alain , Witztum Joseph L , Bruckert Eric , Tselepis Alexandros D , Chapman M John , Tsimikas Sotirios
Published: July 2021

Objectives:
Lipoprotein(a)[Lp(a)] is a causal, genetic risk factor for cardiovascular disease(CVD) and is enriched in pro-inflammatory oxidized phospholipids(OxPL) and lipoprotein-associated phospholipase A2(Lp-PLA2). Epidemiologic outcome studies have shown that the OxPL, Lp(a) and Lp-PLA2 collectively mediate additive risk for CVD.

Methods:
To derive insights into the relationships between Lp(a), OxPL and Lp-PLA2, 18 patients with familial hypercholesterolemia(FH) and Low(<10mg/dl), Intermediate (∼50mg/dl) or High(>100mg/dl) Lp(a) levels on chronic LDL apheresis were evaluated. Twenty-five isopycnic density fractions(density <1.015 to >1.189 g/ml) were isolated from pre and post apheresis plasma(n=50 fractions per patient). OxPL and Lp-PLA2 mass and activity were quantitated with a variety of techniques under 3 conditions: in plasma, in each density fraction with direct plating techniques and on individual lipoproteins within each density fraction by using antibody capture assays to apoB-100(OxPL/apoB and Lp-PLA2/apoB), Lp(a)[OxPL/apo(a) and Lp-PLA2/apo(a)] and apoA-I(OxPL/apoA-I and Lp-PLA2/apoA-I)(total of 900 fractions per measure) and detecting OxPL and Lp-PLA2 with monoclonal antibodies E06 and 4B4, respectively.

Results:
In whole density gradients by direct plating techniques, OxPL were almost exclusively detected in the Lp(a) containing fractions(density 1.059-1.094 g/ml) and Lp-PLA2 mass and activity were present between the apoB and Lp(a) density fractions. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels; Lp-PLA2/apoB and Lp-PLA2/apoA-I levels were highest in patients with Low Lp(a) but decreased proportionally with increasing Lp(a) levels; Lp-PLA2/apo(a) was lowest in patients with Low Lp(a) levels and increased proportionally with increasing Lp(a) levels. LDL apheresis significantly reduces levels of OxPL and Lp-PLA2 on apoB and Lp(a), particularly in patients with high Lp(a) levels

Conclusions:
This study documents that OxPL are primarily present on Lp(a) and Lp-PLA2 primarily on apoB, but the proportion of each increases on Lp(a) and decreases on apoB as plasma Lp(a) levels increase. These data suggest a pathophysiological relationship between Lp(a), OxPL and Lp-PLA2 and provide a rationale for understanding how they collectively mediate CVD in humans.
Abstract 493: Antiatherosclerotic Activity of a New P2y13 Receptor Agonist (ct1007900) in Animal Models
Authors: Barbaras Ronald , Baron Rudi , Goffinet Marine , Tardy Claudine , Boubekeur Nadia , Cholez Guy , Oniciu Daniela C , Lalwani Narendra D , Dasseux Jean-Louis H
Published: July 2021

The F1-ATPase/P2Y13 receptor pathway has been involved in the regulation of the HDL uptake liver and disposition via the reverse cholesterol transport. CT1007900 is a novel selective P2Y13 receptor agonist that has been shown to enhance the HDL uptake that results in the increased secretion of the bile acid, bile cholesterol and bile phospholipid into the gallbladder in mice.

In the present study, CT1007900 has been evaluated in three different animal models of atherosclerosis. In ApoE
-/-
flow cessation model, the administration of the drug decreased the cholesterol concentration in atherosclerotic plaques. In a high fat diet fed ApoE
-/-
mouse model, the prevention of the progression of the plaque in aorta was further evaluated after 4-week treatment with CT1007900. The treated animals had significant decreases in plaque area, cholesterol content, VCAM1 expression and macrophage content.

In the high fat diet fed rabbits, 4-week treatment reduced the cholesterol content by about 30% and also decreased the thickness of the plaques. The ApoA1 mRNA levels in the liver and ApoA1 protein concentration in the plasma increased in the drug-treated animals. The HDL content of the treated animals showed a very consistent pattern with a specific decrease in large HDL and an increase of the “intermediate” size HDL particles as compared to control animals. These intermediate HDL particles seem to be more efficient particles for the removal of cholesterol from atherosclerotic plaques by increasing efflux of cholesterol from the macrophages present in the lesions. The plasma samples from drug treated rabbits showed a dose-dependent increase in the cholesterol efflux in an in vitro assay using J774 cells.

These results clearly demonstrate that improving functionality of HDL rather than the levels of HDL could have a positive impact on the atherosclerotic pathology. These data also support that P2Y13 receptor agonists could be useful pharmacological therapeutics for the treatment of complications due to atherosclerotic disease.
Abstract 523: Acetylation of Cyclophilin A in Response to Oxidative Stress Enhances Its Expression and Secretion
Authors: Soe Nwe Nwe , Sowden Mark , Berk Bradford C
Published: July 2021

Objective:
Cyclophilin A (CyPA) is a Secreted OXidative stress-induced Factor (SOXF) secreted by cardiovascular cells in response to Angiotensin II (Ang II) and reactive oxygen species (ROS). Extracellular CyPA is a proinflammatory mediator that regulates vascular remodeling, abdominal aortic aneurysm, atherosclerosis and cardiac hypertrophy. Post-translational modification of CyPA by acetylation in response to ROS has been described. Moreover, acetylation of CyPA is important in HIV pathogenesis. The mechanism and regulation of CyPA acetylation as well as its role in cardiovascular diseases are currently unknown. We hypothesized that Ang II regulates oxidative stress-induced CyPA acetylation that alters its expression and/or secretion in vascular smooth muscle cells.

Methods and results:
Ang II (1μM) increased acetylation of CyPA (Acyl-CyPA) in a time dependent manner, with a peak at 8hr (3.5±0.6 fold increase) in rat aortic smooth muscle cells (RASMC) as shown by Western blot. Mouse aortic smooth muscle cells from mice lacking CyPA (CyPA-/-) and wild type controls (WT) confirmed that Ang II induced acetylation reactivity coincided exactly with CyPA reactivity. In AT1R and CyPA cotransfected HeLa cells, Ang II increased Acyl-CyPA in a time dependent manner consistent with that in RASMC. The ROS scavengers Tiron or N-acetylcysteine significantly inhibited Ang II induced Acyl-CyPA in a dose dependent manner in RASMC. Ang II-induced CyPA acetylation was enhanced by 2 hr pretreatment with histone deacetylase inhibitor trichostatin (TSA) or sirtinol in a dose dependent manner. Similarly, Ang II-induced CyPA secretion was enhanced by pretreatment with TSA (1μM) in a time dependent manner. Moreover, acetyltransferase p300 and PCAF (p300/CBP-asociated factor) inhibitor anacardic acid (6-nonadecyl salicylic acid) dramatically inhibited CyPA expression, and Ang II induced Acyl-CyPA in a dose dependent manner.

Conclusion:
These results suggest that Ang II-induced CyPA acetylation is oxidative stress dependent, and that acetylation enhanced CyPA expression and secretion. Detailed mechanistic studies of the regulation of CyPA acetylation will help to identify a future therapeutic target for CyPA regulated cardiovascular diseases.
Abstract 527: E2F1 Regulates Syndecan-4 Expression Under Hypoxia
Authors: Biyashev Dauren , Wu Min , Zhou Junlan , Boriboun Chan , Qin Gangjian
Published: July 2021

The E2F1 transcription factor is known to activate the genes involved in cell cycle progression. However, previous results published by our laboratory showed that the E2F1 plays a crucial role in the regulation of in vivo angiogenesis in mouse hind limb ischemia model. More recently, we demonstrated that E2F1-null (E2F1-/-) mice have a significantly better heart function than wild-type (WT) animals after surgically-induced myocardial infarction (MI). E2F1
-/-
mice display reduced endothelial cell (EC) apoptosis, increased EC proliferation, and enhanced angiogenesis in the infarcted heart.

To gain mechanistic insights into the role of E2F1 in modulating cellular response to ischemia/hypoxia, we analyzed global gene expression (Illumina microarray) in WT and E2F1
-/-
mouse embryonic fibroblasts after treatment with hypoxia (0.5% oxygen) or culture under normoxia for 8 h (n=4). The expression patterns of more than 25,000 genes were analyzed.

Among the validated genes syndecan-4, a member of transmembrane proteoglycan family was highly upregulated by hypoxia in E2F1
-/-
cells (p<0.01, n=4). Syndecan-4 is a transmembrane heparan sulfate proteoglycan involved in modulation of cell adhesion, migration, growth factor-mediated signaling, and angiogenesis. It is known to possess pro-angiogenic and anti-apoptotic properties, and in humans the levels of syndecan-4 are drastically increased in the border region of damaged cardiac tissue following MI. In mice, loss of syndecan-4 was shown to increase cardiac cell apoptosis, worsen myocardial remodeling, and provoke cardiac rupture after induced ischemic injury.

We confirmed our findings by measuring the levels of syndecan-4 expression in the hearts of WT and E2F1
-/-
animals after MI. Consistently, we found that the levels of syndecan-4 expression were significantly higher in the E2F1
-/-
mice than in WT mice (p<0.05, n=4).

Our results suggest that there exist a novel mechanism linking E2F1 and syndecan-4 to the regulation of cardiac ischemic angiogenesis.
Abstract 53: Immunoreceptor Tyrosine Activation Motif Signaling in Platelets Is Critical for the Maintenance of Vascular Integrity During Inflammation
Authors: Boulaftali Yacine , Hess Paul R , Cholka Agnieszka , Stolla Moritz , Mackman Nigel , Owens Phil A , Ware Jerry , Kahn Mark L , Bergmeier Wolfgang
Published: July 2021

Introduction:
Blood platelets, long recognized for their importance in primary hemostasis, have recently been identified as critical regulators of vascular integrity during inflammation and cancer. Interestingly, the contribution of platelets to the maintenance of vascular integrity in inflammation seems to be independent of their ability to form a clot.

Goal:
To identify signaling molecules critical for the contribution of platelets to vascular integrity in inflammation.

Methods:
Transgenic mice with platelet-specific expression of the interleukin (IL)4 receptor were rendered thrombocytopenic by infusion of antibodies to IL4R. Platelets from various knockout mice or inhibitor-treated wild-type (WT) platelets were transfused into thrombocytopenic (TP) mice and tested for their ability to support vascular integrity in a reverse passive Arthus reaction (rpA) model. Tissue hemoglobin (Hb) was used to quantify red blood cells in the inflamed tissue, a marker of disrupted vascular integrity.

Results:
Impaired vascular integrity at the site of inflammation was markedly higher in TP mice compared to controls (Hb: 1448±176 vs. 100±8 μg/cm2). Transfusion of WT platelets into TP mice strongly reduced tissue Hb (150±46 μg/ cm2). Transfused platelets from mice lacking the thrombin receptor PAR4 and/or functional P2Y12 (ADP receptor) also prevented disruption of vascular integrity (Hb: 210±66, 136±16 μg and 360±120 μg/cm2), suggesting that signaling by G protein-coupled receptors is not important for this response. Furthermore, infusion of heparin into WT mice did not lead to increased tissue Hb. Immunoreceptor tyrosine activation motif (ITAM) signaling, however, was critical for platelet-dependent maintenance of vascular integrity, as tissue Hb was markedly increased in TP mice transfused with platelets lacking the signal trandsucing adapter protein SLP-76 (1212±160 μg/cm2) or platelets lacking functional ITAM signaling receptors GPVI (852±196 μg/cm2), CLEC-2 (1062±188 μg/cm2), or both (1498±128 μg/cm2). Data are mean±SEM, n=4-5 mice per group.

Conclusions:
We have established a novel mouse model for the adoptive transfer of platelets into TP mice. Using this model we can show that the ITAM but not GPCR signaling in platelets is critical for the maintenance of vascular integrity during inflammation. Our studies highlight a potential clinical complication of future anti-platelet agents targeted towards ITAM signaling receptors such as GPVI.
Abstract 530: Impact of a Single Mixed Mediterranean-Type Meal Compared with a High-Saturated Fat Meal on Postprandial Endothelial Function and Metabolic Markers
Authors: Lacroix Sebastien , Des Rosiers Christine , Gayda Mathieu , Nigam Anil
Published: July 2021

Background:
Endothelial dysfunction is considered a precursor of atherosclerosis and is an independent predictor of cardiovascular events. A high-saturated fat meal (HFM) has been shown to induce postprandial endothelial dysfunction. However, no studies have evaluated the acute endothelial effect of a single mixed Mediterranean-type meal (MMM). Our objective was to evaluate postprandial endothelial and metabolic function in response to a MMM in comparison to an isocaloric HFM.

Methods:
In this ongoing crossover study, 26 of 28 healthy non-smoking males have completed the research protocol. In random order on two separate days during a 1-week interval, subjects were fed two isocaloric meals after an overnight fast. The MMM (885 kcal) consisted of fresh salmon, almonds and vegetables baked in olive oil providing 51% of total calories from fat (7.87g SFA and 2.29g of omega-3, 2:1 DHA:EPA). The HFM consisted of a McDonald’s sausage, egg and cheese McMuffin and three hashbrowns (858 kcal) providing 58% of total energy from fat (14.78g SFA and no omega-3). Endothelial function was evaluated by measuring brachial artery flow-mediated dilation (%FMD) at baseline and at two (T2) and four (T4) hours postprandial.

Results:
Mean postprandial %FMD tends to be less impaired following the MMM than the HFM (variation at T4 -0.15±3.6% vs -2.83±3.3% respectively, p<0.1). Postprandial variations of TG and TG/HDL at T4 were also less severe with the MMM than the HFM (p≤0.05) and did not correlate to %FMD variations. When subdividing the population on the basis of the median fasting TG levels (0.90 mmol/L), the HFM led to significant endothelial impairment in the subjects with higher-TG while it had no effect in the low-TG group.

Conclusion:
Our data suggest that a single MMM exerts less of a deleterious effect on postprandial endothelial function and metabolic markers than does a HFM. A single MMM could thus be less atherogenic than a HFM. Moreover, subjects with higher fasting TG levels (avg. 1.54±0.59 mmol/L, well bellow hypertriglyceridemia threshold) could be at higher risk of endothelial injury following a single HFM.

Data on all 28 subjects will be available in April 2012.
Abstract 536: Prolyl-hydroxylase Inhibition as a Strategy of Therapeutic Angiogenesis
Authors: HoWangYin Kiave-Yune , Loinard Céline , Zouggari Yasmine , Cochain Clément , Récalde Alice , Richart Adèle , Smadja David M , D’Audigier Clément , Duong Van Huyen Jean-Paul , Bruneval Patrick , Emmerich Joseph , Silvestre Jean-Sébastien
Published: July 2021

Therapeutic neovascularization represents an alternative treatment modality for patients with advanced ischemic coronary disease or critical limb ischemia (CLI).

Upregulation of HIF-1α, through specific PHDs inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. The aim of this study was to unravel the efficiency of specific prolyl-hydroxylase domain protein (PHDs) silencing in both cell and non cell-based therapeutic strategies in CLI.

We first showed that PHD2 and PHD3 mRNA levels were up-regulated whereas that of HIF-1α were down-regulated in mononuclear circulating cells and in ischemic legs of diabetic and non diabetic patients with CLI. We then used short hairpin RNAs (shRNAs) to downregulate murine PHD1, PHD2 or PHD3 levels and analyzed their efficiency as a therapeutic strategy in diabetic mice with hindlimb ischemia. PHDs silencing increased post-ischemic neovascularization. This increase was associated with an up-regulation of two key pro-angiogenic factors, VEGF-A and endothelial NOS and with that of pro-arteriogenic factors including fractalkine, MCP-1, and SDF-1 leading to activation of postischemic immunoinflammatory response. In addition, concomitant silencing of PHD2 and HIF-1α abrogated the shPHD2-related effects, demonstrating that activation of HIF-1α signaling mediated the pro-angiogenic and pro-arteriogenic effects of shPHD2. Finally, we assessed the putative beneficial effects of PHDs silencing on human endothelial progenitor cells (hEPC) or bone-marrow derived mesenchymal stem cells (hMSC)-based therapies. Cells were transfected with siRNA directed against PHDs and injected in Nude mice with hind-limb ischemia. We showed that PHDs silencing enhanced human cells therapeutic effect in the ischemic leg of Nude mice compared to animals receiving untreated cells.

In conclusion, our study reveals, for the first time, that silencing of PHDs by the transient and local upregulation of endogenous HIF-1α improves therapeutic angiogenesis. This study also paves the way for future strategy based on administration of siRNAs directed against PHDs to enhance the efficiency of cell-based strategy.
Abstract 538: LDL Particle Core Enrichment in Cholesteryl Oleate Results in Increased Binding to Arterial Wall Proteoglycans and Increased Atherosclerosis
Authors: Melchior John , Kelley Kathryn , Wilson Martha , Sawyer Janet , Hantgan Roy , Rudel Lawrence
Published: July 2021

INTRODUCTION:
Accumulation of lipids in the artery wall, particularly cholesteryl esters (CE), is a classic feature of atherosclerosis. Apo B-containing lipoprotein particles are the primary vehicles by which CEs are delivered across the endothelial barrier into the intima and once present in the subendothelial space these particles are subject to sequestration by native proteoglycans. Several studies in both non-human primate and murine models of atherosclerosis strongly suggest that core enrichment in cholesteryl oleate of low-density lipoprotein (LDL) particles play a significant role in determination of the extent of atherosclerosis. It has also been shown that Acyl-CoA:cholesterol O-acyltransferase 2 (ACAT2) is the enzyme responsible for cholesteryl oleate enrichment of apo B-containing lipoproteins and gene deletion of ACAT2 in animal models is protective against the development of atherosclerosis. Thus, we hypothesized that the selective accumulation of LDL within the intima is the result of LDL particle core enrichment of cholesteryl oleate that results in modification of key surface characteristics of ApoB promoting interaction with resident proteoglycans.

METHODS:
Apo B-100 only, LDLr
-/-
mice (W/T) and Apo B-100 only, LDLr-/-, ACAT2
-/-
(KO) mice were fed diets enriched in either cis-monounsaturated fatty acids (cis-MUFA) or n-3 polyunsaturated fatty acids (n-3 PUFA) for 16 weeks. Blood and plasma was harvested and LDL particles were isolated by size exclusion chromatography. The major lipid constituents of the LDL particle were measured along with the fatty acids of the cholesteryl ester fraction of the particle core. LDL affinity to arterial proteoglycans was determined using an immunocapture surface plasmon resonance (SPR) technique we developed. Atherosclerosis was quantified by measuring the cholesterol content of the aorta.

RESULTS:
W/T mice fed a cis-MUFA diet displayed the highest degree of cholesteryl oleate packaging into the particle core and the highest propensity to bind to arterial proteoglycans. Feeding a diet enriched in n-3 PUFA and/or knocking out ACAT2 successfully inhibited the packaging of cholesteryl oleate into the LDL particles. Accompanying this decrease in cholesteryl oleate content was a significant decrease in binding to arterial proteoglycans and development of atherosclerosis.

CONCLUSION:
Elimination of cholesteryl oleate from the LDL particle core results in significantly less binding to arterial wall proteoglycans and in turn, less development of atherosclerosis.
Abstract 545: Redox-Sensitive Regulation of Bone Marrow Hematopoietic Progenitor Cells
Authors: Pan Shi , Wang Nadan , Sheu Shey-Shing
Published: July 2021

Bone marrow failure is a group of hematopoietic stem cell disorders that affect one or more lineages of blood cells. Hematopoietic progenitor cells are highly sensitive to reactive oxygen species (ROS)-induced oxidative stress. ROS impair the self-renewal of hematopoietic progenitor cells in the bone marrow and cause bone marrow failure. Glutaredoxin (Grx) is an antioxidant enzyme important for many cellular processes such as resistance against oxidative stress, DNA synthesis, sulfur assimilation, apoptosis, and cellular differentiation. However, the role of Grx in hematopoietic progenitor cells remains unknown.

We study the role of Grx in hematopoietic progenitor cells using Grx KO mice as the model system. Bone marrow cells were isolated from 9-10 weeks old wild type and Grx KO mice and the ratio of KSL cells, total blood count and p38 activation was studied. Our results showed no significant difference on the ratio of KSL cells between wild type and Grx KO mice in basal condition. There is also no significant difference in the numbers of red blood cells, lymphocytes, white blood cells and platelet in basal condition between wild type mice and Grx KO. Interestingly, upon hydrogen peroxide treatment, there is higher level of p38 activation in Grx KO mice compared to wild type mice. Our results suggest that Grx is involved in redox-sensitive regulation of hematopoietic progenitor cells. Grx may play a regulatory role in the prevention of bone marrow failure.
Abstract 549: Extracellular Heat Shock Protein 27 Signals Through NF-κB to Favorably Modulate Macrophage Inflammation
Authors: Seibert Tara , Shi Chunhua , Chen Yong-Xiang , Salari Samira , Raizman Joshua , Zhao Xiaoling , O’Brien Edward
Published: July 2021

Background:
Previously we demonstrated that recombinant Heat Shock Protein 27 (rHSP27) reduces atherosclerotic lesion formation when administered to ApoE
-/-
mice. In addition, administration of rHSP27 to ApoE
-/-
mice with established atherosclerotic lesions halts lesion progression and is associated with lesion modifications that are consistent with resilience to plaque rupture. However, the mechanism(s) for these therapeutic effects remain elusive.

Objective:
To determine whether rHSP27 favorably modulates macrophage inflammation by focusing on NF-kB signaling.

Methods/Results:
Activation of the NF-kB signaling pathway by rHSP27 was observed in peritoneal macrophages from ApoE
-/-
mice. Treatment with rHSP27 for 30 minutes activated the translocation of the NF-kB p65 subunit from the cytosol to the nucleus as observed by immunolabeling. A dose-dependant increase in rHSP27 mediated NF-kB activation was observed in RAW 264.7 macrophages stably transfected with an NF-kB inducible reporter gene (15 fold; p<0.05). The use of an N-terminal deletion mutant of rHSP27, rC1, at equimolar concentrations did not induce NF-kB activation, demonstrating specificity of the full-length protein. In addition, NF-kB inhibitors (BAY 11-7082 and MG-132) attenuated the induction of the NF-kB reporter gene by rHSP27, thereby implicating the involvement of IkBa phosphorylation and degradation by the proteasome. A consequence of rHSP27 signaling in macrophages was the up-regulation of the transcript for the haematopoietic growth factor/regulator, GM-CSF (300 fold; p<0.05) and subsequently its secretion (400 fold, p<0.05). In the presence of BAY, GM-CSF expression was inhibited suggesting the involvement of NF-kB.

Conclusions:
rHSP27 promotes the nuclear translocation and activation of NF-kB in macrophages which results in the up-regulation of GM-CSF, a haematopoietic growth factor/regulator that may be responsible for the observed therapeutic effects of rHSP27 in vivo. Current studies are testing rHSP27 therapy in ApoE
-/-
mice deficient in GM-CSF in order to evaluate the importance of macrophage modulation for the beneficial affects of rHSP27 in atherogenesis.
Abstract 75: Heparanase Mediates Arteriothrombosis Following Vascular Injury or Endovascular Stenting
Authors: Baker Aaron , Gibson William , Kolachalama Vijaya , Golomb Mordechai , Indolfi Laura , Spruell Christopher , Zcharia Eyal , Vlodavsky Israel , Edelman Elazer
Published: July 2021

Background:
Heparanase cleaves heparan sulfate proteoglycans (HSPGs), key components of the extracellular matrix and critical endothelial mediators of hemostasis. Our previous work has shown that heparanase levels are elevated in atherosclerosis and following endovascular stenting.

Methods:
We used mice overexpressing human heparanase and examined the time to thrombosis using a laser induced arterial thrombosis model in combination with vascular injury. An ex-vivo system was used to examine the formation of thrombus to stent-induced flow disturbance. To evaluate thrombotic potential in wild-type (WT) or heparanase transgenic (HPA-Tg) mice, the left common carotid artery was isolated and a vascular flow probe was used to monitor blood flow. Rose Bengal dye was injected into the mice and the mid portion of the common carotid artery was then illuminated with a 1.5-mW green laser (540 nm) until an occlusive thrombus was formed. Arterial injury was also performed prior to measuring thrombosis to examine the effects of light and heavy injury on thrombosis in WT and HPA-Tg mice. A novel ex-vivo assay of stent thrombosis was performed in which mouse aortas were stented into a modified Chandler loop and the loops were perfused with blood from transgenic mice. Results were analyzed with electron microscopy and optical absorbance for clot formation.

Results:
In the absence of injury there was no difference in the time for formation of thrombus in the WT and HPA-Tg mice. However in the presence of arterial injury, thrombosis in HPA-Tg mice occurred significantly faster than in WT mice (light injury: 67.7 minutes vs. 39.8 minutes, p<0.05; heavy injury: 63.0 minutes vs. 41.8 minutes, p<0.05). The prothrombotic phenotype of heparanase transgenic mice is masked when heparin is administered prior to injury (WT: 68.5 minutes vs. HPA Tg: 69.2 minutes p=NS). Blood from heparanase transgenic mice also increased stent thrombosis threefold in flow loops as measured by optical density (2.8 vs. 1.0 optical density, p<0.05) and increased adherent platelets were found in electron microscopy.

Conclusions:
Increased arterial expression of heparanase leads to enhanced risk of thrombosis in the injured artery. In particular, endovascular stenting induces both injury and flow disturbance that exacerbate the thrombotic potential of blood with increased heparanase levels.