bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 103: Biomechanical Rupture Risk Assessment in Patients with Abdominal Aortic Aneurysms: Introducing Rupture Risk Equivalent Diameter
Authors: Roy Joy , Swedenborg Jesper , Sakalihasan Natzi , Nchimi Alain , Bockler Dittmar , Hyhlik-Durr Alexander , Gasser Christian
Published: March 2022

OBJECTIVES:
Finite Element (FE) Analysis has been used to estimate Peak Wall Stress (PWS) and Peak Wall Rupture Risk (PWRR) of Abdominal Aortic Aneurysms (AAA). However, these values are not familiar for clinicians. The aim of this study was to introduce a patient specific and clinically applicable biomechanical rupture risk assessment tool that would be easy for Vascular Surgeons to comprehend.

METHODS:
Clinical data(gender, age, smoking, chronic obstructive lung disease, mean arterial pressure, family history) and CT images were retrospectively gathered from 200(142 male, 44 female) non ruptured AAA patients from 4 different hospitals in Sweden, Belgium and Germany. FE models were created using the diagnostics system A4clinics (VASCOPS, Austria) and the maximum diameter, PWS and PWRR was calculated automatically. Statistical analysis was performed with Mathematica (Wolfram Research Inc, USA).

RESULTS:
The maximum diameter was normally distributed in males and females and no difference was found between PWS levels in men and women (p=0.091) but the PWRR was higher in women (p=0.005). PWS increased in a linear fashion and PWRR exponentially with diameter. We then related PWRR to the maximum diameters of patients and calculated the Rupture Risk Equvialent Diameter (RRED) as shown in the figure. A PWRR of 0.48 corresponds to an RRED of 55 millimeters. We also found that a RRED of 55 mm corresponds to the rupture risk in a male with a maximum AAA diameter of 58 mm, but in a female the corresponding maximum diameter was only 46mm.

CONCLUSIONS:
Biomechanical AAA rupture risk assessment integrates risk based on clinical parameters and data from CT images. The RRED expresses this information as a diameter that is comprehensible for clinicians.
Abstract 107: Protein Comigration Pattern Analysis to Identify High-Density Lipoprotein Subspecies
Authors: Deng Jingyuan , Gordon Scott , Davidson Sean , Long Lu
Published: March 2022

High density lipoproteins (HDL) are micromolecular complexes composed of lipids and proteins that play critical roles in the prevention of cardiovascular disease (CVD), the major cause of mortality in the United States. Recent proteomics studies have identified about 75 distinct HDL-associated proteins. Many of the newly identified proteins mediate functions that are surprisingly outside the realm of lipid transport. Recently there is strong evidence suggesting that diverse HDL functions are mediated by distinct HDL subspecies. To study the HDL subspecies and their associated functions in CVD, we have applied three orthogonal separation chromatography techniques (gel filtration, anion exchange and isoelectric focusing) to fractionate normal human plasma to phospholipid-containing subfractions. We then isolated lipid associated proteins and determined their identities and relative distribution across subfractions using mass spectrometery. The distribution patterns of 159 proteins were determined for each separation technique. Lipid associated proteins which have similar co-migration patterns when separated by different techniques are likely to form distinct lipoprotein subspecies. Two novel correlation based score systems were developed in order to identify these subspecies: the C-Score and S-Score systems. C-Score system is based on the number of fractions that contain both proteins. S-Score system is based on the variation of two proteins’ normalized patterns, which does not require the independence assumption required by regular correlation coefficient. We built two similarity networks based on these two score systems and identified 31 overlapped cliques. These 31 identified cliques may represent consistent HDL subspecies across different human plasma samples and analysis methods. To assess the significance of these cliques, we performed random permutation test. We further did functional enrichment analysis using GO annotations. All of these analyses suggest that these candidate cliques/biomarker may reveal novel interactions among HDL proteins and provide models of the underlying molecular mechanisms of CVD. The experimental validations of the identified HDL subspecies using immunoprecipitation are under way.
Abstract 111: Effects of the Novel Factor Xa Inhibitor, Apixaban, on Elevated Levels of Thrombotic Biomarkers in Genetically Obese, Diabetic Mice
Authors: Arey Brian J , Bird Eileen , Seiffert Dietmar , Knabb Robert
Published: March 2022

Obesity and related sequelae such as insulin resistance and diabetes present a growing health care burden throughout the world. One of the hallmarks of the disease process in these patients is the presence of a prothrombotic state that manifests itself as heightened levels of circulating thrombotic/platelet biomarkers such as thrombin-antithrombin complex (T-AT), soluble CD40 ligand (sCD40L) and inflammatory cytokines such as interleukin 6 (IL6). We have found that levels of these biomarkers are also elevated in genetic models of obesity and diabetes (db/db mice). Apixaban is a potent, highly selective, direct Factor Xa inhibitor that shows great promise as a safer and more efficacious therapy then the existing standard of care (ARISTOTLE trial). For this reason, we sought to study the differences in the ability of apixaban and current clinical therapies to lower plasma markers of thrombosis (T-AT and sCD40L) in diabetic mice. We administered either apixaban, warfarin or aspirin and clopidogrel to 12 week old, male
db/db
mice p.o. for 48 h prior to collection of blood. T-AT and sCD40L were measured in plasma by ELISA.
db/db
mice had 3-fold greater levels of T-AT (P<0.05) and 2-fold (P<0.05) greater levels of sCD40L in plasma as compared to C57Bl6 mice. Warfarin (0.3 mg/kg) dose-dependently decreased T-AT (P<0.05) to levels similar to normoglycemic C57/Bl6 controls. Apixaban (5 and 50 mg/kg) significantly reduced (P<0.05) elevated levels of these biomarkers when administered b.i.d. When administered q.d., apixaban (10 and 100 mg/kg) lowered T-AT levels but did not robustly affect sCD40L. The platelet therapies, aspirin (30 mg/kg) and clopidogrel (3 mg/kg) robustly decreased sCD40L (P<0.05) but did not affect T-AT levels in obese, diabetic mice. Overall, these data suggest that administration of apixaban is equally efficacious as warfarin in lowering elevated thrombotic biomarkers in diabetic mice. Anti-platelet therapies were efficacious on platelet biomarkers but did not affect markers associated with elevated thrombin activation. This study was funded by Bristol-Myers Squibb and Pfizer.
Abstract 112: Reconstitution with Saline Is More Appropriate than Arithmetic Correction When Examining Stress-Hemoconcentration Effects on Coagulation
Authors: Austin Anthony W , Patterson Stephen M
Published: March 2022

Introduction:
The goal of this study was to examine a new physiologically relevant method of correcting for hemoconcentration effects on stress-induced changes in coagulation parameters (i.e., activated partial thromboplastin time (APTT), prothrombin time (PT%), FVIII:C, and FVII:C). Hypotheses: We hypothesized that 1) Area-under-the-curve (AUC) for uncorrected PT% and FVII:C would be greater than AUC corrected arithmetically or with saline or plasma, 2) AUC for APTT uncorrected or corrected with baseline plasma would be less than AUC when corrected with saline and greater than AUC corrected arithmetically, and 3) AUC for FVIII:C would not differ between correction methods. Methods: Blood was drawn from 40 men during a 20-min baseline, a 6-min math stressor and a 20-min recovery period. Two reconstitution techniques were used to adjust for hemoconcentration during stress and recovery using the person’s own plasma from baseline or physiological saline equal to the amount of plasma lost during each period. Results: For PT%, uncorrected AUC (M = 30.6 ± 18.4) was significantly greater than AUC corrected with saline (M = -33.3 ± 25.5, p = .002) or arithmetically (M = -9.1 ± 20.3, p = .001) but was not significantly different from AUC corrected with plasma (M = 25.5 ± 20.8, p = .72). For FVII:C, uncorrected AUC (M = 21.0 ± 33.1) was significantly greater than AUC corrected arithmetically (M = -31.7 ± 36.1, p = .001), but was not significantly different from AUC corrected with saline (M = -6.4 ± 38.3, p = .17) or plasma (M = 33.8 ± 36.8, p = .47). For APPT, uncorrected AUC (M = -15.2 ± 4.9) was significantly less than AUC corrected with saline (M = -6.3 ± 5.2, p = .001) and greater than AUC corrected arithmetically (M = -26.7 ± 6.8, p = .007), but was not significantly different from AUC corrected with plasma (M = -13.5 ± 5.2, p = .47). For FVIII:C, the omnibus test was non-significant (p = .39), suggesting no differences between groups. Conclusions: Saline reconstitution appears to be a more biologically relevant correction method when examining hemoconcentration effects on clotting time and clotting activity, whereas the plasma reconstitution method and arithmetic correction do not seem appropriate. With the exception of FVIII:C, hemoconcentration appears to account for the changes in the coagulation parameters.
Abstract 117: P53 Silenced Endothelial Progenitor Stem Cells (EPC) Improve Collateral Circulation Post--Femoral Artery Occlusion in Diabetic Mice
Authors: Sen Sabyasachi , Young Mary , Chou Cyril , Jerry Joseph
Published: March 2022

Literature shows that EPCs contribute to increased collateral vessel formation following vaso-occlusion. However diabetes reduces EPC number and reduces collateral vessel formation. We cultured human EPCs and exposed them to 5.5 mM (equivalent to 99mg%) and 20mM (equivalent to 360mg%, HG) glucose, which resulted in significant EPC death within 48hrs. We noted HG exposure was associated with up-regulation of P53 and its downstream genes such as P21, PUMA and Caspase-3. We hypothesized that EPC reduction in hyperglycemia is secondary to up-regulation of pro-apoptotic gene, P53. We obtained mouse peripheral blood derived EPCs from P53 null mice and observed that p53 KO EPCs are more resistant to cell death in HG. P53 null EPCs evolved into mature mouse EC (MEC) and retained endothelial properties such as cobblestone appearance and tube-formation on matrigel. Next, we used Lenti and Adenovirus mediated si-RNA methods to silence P53 (long and short term respectively) in human EPCs and P53 silenced EPc showed better survival in HG. We developed femoral artery occlusion model to mimic peripheral vascular disease in diabetic animals. We used streptozotocin induced type 1 diabetic or Leptin resistant type 2 diabetic mouse model. We delivered saline, P53 WT and P53 null EPCs intra-muscularly around the femoral occlusion in these mouse models (n=10 in each group) and counted CD31+ve number of capillaries in a 20x microscopic field. Highest number was noted in the group that received P53null EPCs ( 26.3±4.2, 49.6±7.4 and 95.7±9.7 respectively). Summary: Transplantation of P53 null EPCs in hyperglycemic mice with femoral occlusion demonstrate better collateral vessel formation post femoral artery occlusion setting compared to WT-EPC transplanted group. Conclusion: This finding indicates towards possible therapeutic benefit in transplanting short-term P53 silenced human EPC to prevent peripheral vascular disease in patients with diabetes.
Abstract 118: Protein Kinase C-delta Prompts Adventitial Cell Migration Through Regulation of Monocyte Chemoattractant Protein-1 Signaling Pathway in Intimal Hyperplasia
Authors: Si Yi , Ren Jun , Shi Xudong , Kent K Craig , Liu Bo
Published: March 2022

Introduction:
Increasing evidence indicates that adventitial cells directly contribute to intimal hyperplasia via migration. Protein kinase C delta (PKC δ), a master mediator of apoptosis of smooth muscle cells (SMCs), is known to mediate expression of monocyte chemoattractant protein-1(MCP-1). In the current study, we hypothesize that PKCδ promotes adventitial cells migration by regulation of MCP-1 signaling pathway in injured arteries.

Methods and results:
Using isolated carotid SMCs and adventitial fibroblasts, we showed that conditioned media from PKCδ-overexpressing SMCs stimulated migration of adventitial cells, which was blocked by neutralizing MCP-1 or by CCR2 mRNA knocking down. In vivo, PKCδ gene transfer increased SMC apoptosis and levels of MCP-1 and CCR2 expression (MCP-1 protein measured by immunostaining in pixel: 397±20*103 vs 99±18*103, p<0.05; CCR2 mRNA fold induction: 3.45±0.09 vs 1, p<0.01). Next, we evaluated adventitia cells migration by labeling adventitial cells with AdLacZ administered to the adventitial surface immediately following the carotid angioplasty procedure. Overexpresison of PKCδ in SMCs increased the accumulation of adventitial cells in the neointima by close to 10 times (LacZ positive area in pixel of AdPKCd- and AdNull-infected arteries: 769±67*103 vs 84±11*103, p<0.01). Blocking the MCP-1 signaling with an anti-MCP-1 antibody dramatically diminished the number of adventitia-originated cells detected in the medial/intimal region (LacZ positive area in pixel: 357±37*103 vs 894±42*103, p<0.01). The joint treatment of PKCδ and anti-MCP-1 markedly inhibited the I/M ratio by 62% as compared to a 36.3% and 30.8% reduction induced by a single treatment of PKCδ and anti-MCP-1, respectively. None of the treatments altered macrophage infiltration.

Conclusions:
PKCδ appears to play multiple functions in arterial injury response. Apart from inducing apoptosis, PKCδ regulates migration of adventitial cells via upregulation of the MCP-1/CCR2 signaling axis. A combined strategy targeting both SMC apoptosis and adventitial cell migration may be an efficient approach to inhibit intimal hyperplasia.
Abstract 133: Nicotine Treatment Changes the [18F]FDG and [18F]2FA Uptake Level to Cultured Macrophages
Authors: Ogawa Mikako , Kosugi Mutsumi , Suzuma Kyotaro , Magata Yasuhiro
Published: March 2022

Objectives:
It is well known that smoking is one of the risk factors for atherosclerosis, and previous reports have shown that nicotine induces cytokine release in cultured macrophages and stimulates plaque growth in a mouse model of atherosclerosis. We and other group have shown that a glucose metabolism imaging probe for PET, [
18
F]FDG, can specifically detect vulnerable plaque depending on the macrophage infiltration extent. In this study, we investigated the effects of nicotine on [
18
F]FDG uptake in cultured macrophages. In addition, the uptake study was also performed with α4β2 nicotine receptor imaging agent, [
18
F]2FA.

Methods:
Macrophages were isolated from mice peritoneum following thioglycollate injection, and cultured for 24hrs. Then, nicotine was added to each culture well at the concentration of 0, 0.1, 1 or 10 μM. After the culture for 24 hrs, [
18
F]FDG or [
18
F]2FA was added to each well and incubated for 1hr at 37°C. Then, the cells were peeled from each well and the radioactivity was measured. The protein concentration was measured by Lowry’s method.

Results:
[
18
F]FDG uptake to the cells were 5.6 ± 0.8, 7.7 ± 2.3, 8.1 ± 1.8 and 8.7 ± 2.3 %dose/mg protein at 0, 0.1, 1 and 10 μM nicotine, respectively. The significantly higher uptake was observed in 1 and 10 μM nicotine treated groups compared to untreated group (0μM). [
18
F]2FA uptake was higher in 10μM nicotine treated group than untreated group.

Conclusions:
Our results show that macrophage metabolic activity was activated by nicotine, and nicotine α4β2 receptor up-regulation was induced by nicotine treatments. Further investigation with α7 nicotinic receptor imaging agent is undergoing to elucidate the relationship between nicotinic receptor and atherosclerosis.
Abstract 138: Deficiency of CD11a Reduces CD8+ T-Cell Activation and Proliferation in Adipose Tissue of Obese Mice
Authors: Jiang Erlie , Dai Perrard Xiaoyuan , L Perrard Jerry , Smith C Wayne , M Ballantyne Christie , Wu Huaizhu
Published: March 2022

T cells, particularly CD8
+
T cells, are major participants in obesity-linked adipose tissue (AT) inflammation. CD11a, a β
2
-integrin, is crucial for T cell adhesion and interactions with antigen-presenting cells. It has been shown that stimulation of CD11a lowered the threshold of T cell activation, yet the role of CD11a in T cell activation in AT remains unknown. We used CD11a
-/-
mice to test the hypothesis that CD11a deficiency reduces CD8
+
T cell activation in AT. Mice fed high-fat diet for 3 months were used as an obesity model, with mice on normal diet as lean controls. CD8
+
T cells and related cytokines were examined in AT by flow cytometry or quantitative RT-PCR. Compared to lean wild-type (WT) mice, obese WT mice had increased numbers of activated CD8
+
T cells, with higher proportions of CD11a
high
CD8
+
memory T cells, CD44
+
CD62L
-
effector memory CD8
+
T cells and CD44
+
CD69
+
effector CD8
+
T cells; and increased mRNA levels of granzyme B and IFN-γ in AT. Compared to obese WT, obese CD11a
-/-
mice had lower proportions of CD44
+
CD62L
-
effector memory CD8
+
T cells and CD44
+
CD69
+
effector CD8
+
T cells, and decreased mRNA levels of granzyme B and IFN-γ (P<0.01), implying decreased activation of CD8
+
T cells. IL-2, IL-12 and IL-18 levels were increased in AT of obese WT compared to that of leans, and were lower in AT of obese CD11a
-/-
mice than that of obese WT. In vitro treatment with IL-2, IL-12 and IL-18 induced proliferation and expression of IFN-γ and CD69 in CD8
+
cells isolated from AT of WT mice. Compared to those from WT mice, CD8
+
T cells from AT of CD11a
-/-
mice showed decreased response to cytokine stimulation with fewer cells expressing IFN-γ (7.0±1.1% in CD11a
-/-
vs. 11.3±1.1% in WT, n=3-5/group, P<0.05). In addition, using Edu labeling techniques, we studied proliferation of CD8
+
T cells in AT in vivo. At 3 hours after intraperitoneal injection of Edu, no Edu
+
T cells appeared in blood but we found CD8
+
/Edu
+
T cells in AT of WT mice, indicating CD8
+
T cells proliferated in AT. Compared to obese WT mice, obese CD11a
-/-
mice had decreased proportion of proliferating CD8
+
T cells in AT (1.3±0.4% in CD11a
-/-
vs. 2.4±0.1% in WT, n=3/group, P<0.01). In conclusion, we demonstrated that CD11a played a crucial role in CD8
+
T cell activation and proliferation in AT associated with obesity.
Abstract 139: Id3 Regulation of High-Fat-Diet--Induced Visceral Adipose Tissue Macrophages
Authors: Kaplan Jennifer L , Garmey James C , Harmon Daniel B , Oldham Stephanie N , McNamara Coleen A
Published: March 2022

BACKGROUND:
Global deletion of the helix-loop-helix (HLH) transcription factor Inhibitor of Differentiation 3 (Id3), attenuates HFD-induced obesity. Specifically, Id3
-/-
mice have less visceral adipose tissue (VAT); an effect that may be linked to VAT inflammation. Adipose tissue macrophages (ATMs) play a key role in HFD-induced obesity. Monocyte chemoattractant protein-1 (MCP-1) is a key cytokine regulating VAT macrophage accumulation, yet the role of Id3 in regulating MCP-1 expression and ATM accumulation is unknown.

RESULTS:
Id3
-/-
mice and C57Bl/6 litter-mate controls were fed 4 weeks of HFD. The epididymal adipose tissue (VAT) and sera were analyzed for MCP-1 expression. Consistent with published findings, results demonstrated that HFD induced MCP-1 mRNA (analyzed via qPCR) and protein expression (analyzed via ELISA) in VAT. Notably, there was marked attenuation of HFD-induced MCP-1 expression in Id3
-/-
mice. Epididymal adipose tissue from both WT and knockout mice were cultured with and without IL-1β. MCP-1 protein levels were the same in both sets of mice, when fat from chow-fed mice was cultured in media alone. However, IL-1β induced MCP-1 protein expression in VAT, an effect significantly attenuated in mice null for Id3. Flow cytometry analysis revealed that at early time-points (4 weeks HFD), WT mice have a significant increase in M1 macrophage numbers in epididymal VAT in response to HFD. However, Id3 null mice had an attenuation of this response to HFD. In contrast, HFD induced a statistically significant increase in M2 macrophages in both WT and Id3
-/-
mice. RAW 264.7 cells were stimulated with 100ng/ml of LPS for 0, 0.5, 3, 6 and 24 hours. LPS treatment led to a significant and rapid induction of Id3 mRNA expression, which preceded upregulation of MCP-1 mRNA and protein levels. NIH 3T3 cells were co-transfected with a 1-kb MCP-1 luciferase promoter construct, as well as an expression plasmid encoding E12, a member of the bHLH E-protein family of transcription factors. Increasing concentrations of E12 led to repression of MCP-1 promoter activity, seen as a decrease in RLU/s compared to transfection with the promoter alone..

CONCLUSION:
The loss of Id3 decreases local and systemic MCP-1 expression, and decreases M1 macrophage numbers in visceral adipose tissue. Id3’s partner E12 represses MCP-1 promoter activity. Based on the Id3-E-protein paradigm, it is likely that Id3 would restore MCP-1 promoter activity. These data suggest that Id3 promotes HFD-induced MCP-1 expression and macrophage-mediated visceral adipose tissue expansion. Further studies are needed to confirm Id3 regulation of MCP-1 specifically in macrophages.
Abstract 140: LRP6 Influences Body Fat and Glucose Homeostasis in Mouse by Activating mTOR Pathway and Inhibiting Mitochondrial Energy Expenditure
Authors: Liu Wenzhong , Singh Rajvir , Choi Cheol Soo , Young Lee Hui , Keramati Ali R , Samuel Varman T , Lifton Richard P , Shulman Gerald I , Mani Arya
Published: March 2022

Background:
Body fat, insulin resistance and type2 diabetes are often linked together. Wnt signaling regulates adipogenesis and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. Mutations in Wnt coreceptor LRP6 have been associated with diabetes, coronary heart disease and metabolic syndrome in humans, but the molecular mechanisms are largely unknown.

Methods:
We study the role of LRP6 in regulation of glucose and energy metabolism in heterozygote LRP6 knockout mice on high fat diet by using glucose tolerance test, hyperinsulinemic-euglycemic clamp studies and dissection of insulin signaling pathway.

Results:
LRP6+/- mice on high fat diet were protected against diet -induced obesity and hepatic and adipose tissue insulin resistance compared to wildtype littermates. Brown adipose tissue insulin sensitivity and lean body of LRP
+/-
mice were caused by diminished Wnt -dependent mTORC1/S6K activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6
+/-
mice also exhibited reduced endogenous hepatic glucose output which was due to diminished FoxO1-dependent activity of G6Pase. In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with tissue specific increase of leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6
+/-
mice.

Conclusion:
Our study identifies LRP6 as a nutrient-sensitive regulator of the body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.
Abstract 143: Group X Secretory Phospholipase A2 Regulates Insulin Secretion by Mouse Pancreatic Beta Cells
Authors: Shridas Preetha , Zahoor Lubna , Bailey William , Forrest Kathy , Webb Nancy R
Published: March 2022

Background:
Group X secretory phospholipase A2 (GX sPLA2) potently releases arachidonic acid (AA) from the plasma membrane of intact cells. AA is a precursor of bioactive prostaglandins that are known to modulate insulin secretion by β-islet cells. C57BL/6 mice deficient in GX sPLA2 (GX KO) are protected from age-related defect in glucose tolerance. GX sPLA2 is expressed in mouse pancreatic islet cells. In this study we tested the hypothesis that GX sPLA2 regulates pancreatic insulin secretion.

Methods and results:
Glucose stimulated insulin secretion (GSIS) was measured in vivo in WT and GX KO mice and ex vivo using pancreatic islet cells isolated from WT and GX KO mice. To complement these studies, GSIS was also assessed in vitro using Min6 pancreatic beta cell lines with or without GX sPLA2 overexpression. GSIS was significantly increased in GX KO mice compared to WT mice, and in islet cells isolated from GX KO mice compared to WT mice. Consistent with this finding, Min6 cells overexpressing GX sPLA2 demonstrated significantly decreased GSIS compared to control cells. Expression of ABCA1 and ABCG1 mRNAs were significantly upregulated in islet cells from GX KO mice compared to WT mice, consistent with our previous report that GX sPLA2 negatively regulates LXR activity. However, there was no significant difference in cholesterol content between islets from WT and GX KO mice suggesting that altered LXR activity may not be the mechanism. Min6 cells overexpressing GX sPLA2 secrete significantly increased levels of PGE2- a known negative regulator of GSIS, in the medium compared to control cells. Treatment of these cells with NS398- an inhibitor of COX2 enzyme, abolished the decrease in GSIS shown by these cells.

Conclusions:
We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion most likely by enhancing PGE2 production.
Abstract 151: Role of Hypoxia Inducible Factor-1α in Leukocyte Recruitment to Sites of Vascular Injury and Atherosclerotic Progression in ApoE-/SRB - mice
Authors: Dong Feng , Khalil Mazen , Anwaruddin Saif , Penn Marc S
Published: March 2022

Introduction:
HIF-1α is an inducible protein stabilized by hypoxia and is thought to be an important mediator of leukocyte metabolism and inflammation. The role of HIF-1α in acute coronary syndrome (ACS) remains undefined. Since HIF-1α is a lethal knockout, we generated ApoE/SRB double null (DN) mice with HIF-1α null leukocytes using bone marrow transplantation (BMT) and hematopoietic stem cells (HSC) transducted with a lentivirus encoding a siRNA to HIF-1α or scramble.

Methods:
Donor bone marrow was harvested from 6 week-old ApoE/SRB DN mice. At age 6 weeks, following lethal irradiation ApoE/SRB DN mice received 50,000 scramble or HIF-1 α: siRNA transfected HSC. Mice were weaned from probucol 2 weeks after BMT. Serial echocardiography was used to assess cardiac function. Burden of coronary atherosclerosis and macrophage recruitment were assessed.

Results:
White blood cells from HIF-1 α: siRNA mice exhibited significantly less HIF-1α expression following culture in hypoxic conditions (p<0.05). HIF-1 α: siRNA mice demonstrated significantly less coronary atherosclerosis (p <0.05) and macrophage recruitment (p=0.02) in the plaque. HIF-1 α: siRNA mice showed significantly delayed time to myocardial infarction. Six weeks after cessation of probucol in the diet, control animals had an ejection fraction of 43 ± 6% compared to 73 ± 7% in HIF-1 α: siRNA mice. HIF-1 α: siRNA mice did not exhibit evidence of left ventricular dysfunction until 7.9 ± 0.4 weeks after weaning off probucol as compared to 5.3 ± 0.3 weeks in the controls. Consistent with delayed onset of myocardial infarction in HIF-1α: siRNA transplanted mice, the mice with decreased HIF-1α expression survived longer than control mice (8.6 ± 0.4 vs. 6.3 ± 0.3, p<0.05).

Conclusion:
Leukocyte derived HIF-1 α plays a critical role in leukocyte recruitment to the site of vascular injury and appears to be involved in the progression of coronary atherosclerosis.
Abstract 172: Characterization and Comparison of High-Density Lipoprotein Subclasses Among Inbred Strains of Mice
Authors: Srivastava Ujala , Kamir Daniela , Deng Bin , Korstanje Ron
Published: March 2022

Aim:
Measurement of HDL subclasses provides insight into lipoprotein metabolism and helps predict risk for cardiovascular disease (CVD). HDL subclasses have been studied using biochemical tools in humans, but extensive studies on the role of HDL subclasses and their protein compositions in HDL metabolism have not been conducted using an animal model. In this study, we established a method to identify HDL subclasses in mice and compared HDL subclasses and the protein cargoes associated with them in three inbred strains of mice.

Methods:
We used a chemical precipitation method described by Warnick et al. in 1982, to identify HDL subclasses. In this method, LDL and VLDL are precipitated out from the serum using dextran sulfate and magnesium chloride solutions. Subsequently, HDL subclass distribution on gels was compared among male mice from 3 inbred strains with low (CAST/EiJ), normal (C57BL/6J) and high (NZB/BINJ) levels of HDL cholesterol (HDL-C). HDL subclasses were visualised by Coomassie Staining and Mass Spectrometry (MS) was done to confirm the presence of ApoA1, a constituent protein of HDL.

Results:
We observed that CAST/EiJ and NZB/BINJ had significantly different patterns of distribution of HDL subclasses compared to C57BL/6J mice. Furthermore, HDL subclasses from C57BL/6J and NZB/BINJ mice were subjected to quantitative MS. 106 proteins were associated with HDL particles in both strains, 47 proteins were unique to C57BL/6J HDL particles and 7 were unique to NZB/BINJ HDL particles. Using the Ingenuity Pathways Analysis we found that 30 molecules (27%) in NZB/BINJ and 33 (22%) in C57BL/6J mice had well characterized roles in lipid metabolism. 22 proteins that were previously identified as HDL associated in humans were identified in mice as well and 38 proteins were identified to be associated exclusively with mouse HDL in this study.

Conclusion:
Protein cargoes associated with HDL define its biological function. Thus, differences in HDL subclasses and HDL-associated proteins could potentially affect HDL function in these mouse-inbred strains, which ultimately could influence the development of CVD.

Figure:
HDL subclasses present in 10-week old C57BL/6J mice (gel was stained with Coomassie Dye to visualize the subclasses; MS was done to confirm presence of ApoA1, a constituent protein of HDL)
Abstract 173: Cathepsin B Is a Novel Gender-Dependent Modifier of Dietary Cholesterol and Fat Absorption from the Intestine
Authors: Wong Winifred P , Schweitzer Jessica B , Hester James F , Chan Ernest R , Serre David , Sehayek Ephraim
Published: March 2022

The absorption of dietary cholesterol and fat from the intestine is an important determinant of major risk factors for atherosclerotic cardiovascular diseases including hypercholesterolemia, obesity, metabolic syndrome, and type 2 diabetes. Nevertheless, the molecular mechanisms regulating the absorption process are only partially understood. We used two inbred mouse strains, C57BL/6J and CASA/Rk that differ in dietary cholesterol and fat absorption, to map a locus on chromosome 14 that modifies the absorption process. Studies in congenics of this locus revealed a complex effect of several genetic determinants. Fine mapping of one genetic determinant allowed identification of a critical 6.3Mb CASA/Rk interval (63.3-69.7 Mb) with gender dependent effect. Therefore, congenic females of the critical interval, but not congenic males, displayed a 30% decrease in dietary cholesterol absorption. RNA-seq studies in jejuna of congenic females found 50% suppression of Cathepsin B (Ctsb), a gene that maps within the critical congenic interval and encodes for a ubiquitously expressed lysosomal cysteine protease. Concordant with findings in congenic females, Ctsb knockout females, but not knockout males, displayed significantly (25%) decreased dietary cholesterol absorption rates and a large (80%) decrease in dietary fat absorption. These studies indicate that: (i) Cathepsin B is a novel gender-dependent determinant of cholesterol and fat absorption from the intestine, and (ii) the critical chromosome 14 interval is likely to harbor additional genetic determinants of cholesterol and fat absorption from the intestine.
Abstract 177: The Effects of Inflammation, Hyperglycemia and Cyclic Strain on Osteoprotegerin Production in Human Aortic Smooth Muscle Cells
Authors: Davenport Colin , Martin Fiona A , Rochfort Keith D , Murphy Ronan P , Smith Diarmuid , Cummins Philip M
Published: March 2022

Introduction:
A pivotal step in vascular calcification is the transformation of smooth muscle cells (SMCs) to osteoblast-like cells. Osteoprotegerin (OPG), a soluble protein produced by SMCs, inhibits this transformation and may represent a therapeutic target for calcification reduction. Hyperglycemia, inflammation and increased levels of cyclic strain are all important inducers of calcification in vivo. The purpose of this study was to determine the effects of these stimuli, both alone and in combination, on OPG production by human aortic SMCs (HASMCs) in vitro.

Methods:
HASMCs were initially exposed to a single stimulus, with OPG levels in cell-conditioned media measured at 24 and 48 hours by ELISA (n=6 for all experiments). Equibiaxial cyclic strain was produced using a Flexercell® Tension-Plus FX-4000T system (0 or 12.5%), whilst hyperglycemic and inflammatory conditions were induced using glucose (6, 15 and 30 mmol/l) and tissue necrosis factor alpha (TNFα, 10 and 100 ng/ml) respectively. HASMCs were then exposed to a combination of hyperglycemia (15 mmol/l), inflammation (10 ng/ml TNFα) and strain (10%), after which levels of OPG and runt-related transcription factor 2 (RUNX2, a marker of osteoblastic transformation) were assessed via ELISA and qRT-PCR, respectively.

Results:
High levels of cyclic strain (12.5%) significantly increased OPG levels in the cell-conditioned media at 24 and 48 hours. OPG levels were also dose-dependently increased by treatment with TNFα at 24 and 48 hours. At 24 hours with 15 and 30mmol/l glucose, there was no change in OPG levels, but at 48 hours increased OPG levels were observed at both concentrations. In the combination experiment, OPG levels decreased, while RUNX2 mRNA levels increased, after 48 hrs.

Conclusions:
Cyclic strain, hyperglycemia, and TNFα increased OPG release by HASMCs when administered in isolation. Applied in combination however, these stimuli decreased OPG levels in parallel with an increase in a marker of osteoblastic transformation. This data suggests that inhibition of vascular calcification by OPG may be actively down-regulated when multiple pro-calcification stimuli co-exist, and supports further investigation of OPG as a therapeutic target for calcification reduction.
Abstract 181: Enhanced Cell Death by Oxidized Phospholipids in Vascular Smooth Muscle Cells via Activation of AMP-Activated Protein Kinase
Authors: Greig Fiona H , Ewart Marie-Ann , Spickett Corinne M , Kennedy Simon
Published: March 2022

The modification of phospholipids, principally found in low density lipoproteins, is thought to be a primary risk factor in the progression of atherosclerosis. AMP-activated protein kinase (AMPK) is involved in regulating cellular energy balance and believed to be vasculoprotective in many cardiovascular diseases including atherosclerosis. This study investigates the interaction of modified phospholipids on AMPK function in vascular smooth muscle. Aortic smooth muscle cells (SMCs) were isolated from male New Zealand white rabbits and incubated with increasing concentrations of oxidized phospholipids, 1-palmitoyl-2-glutaroyl-
sn
-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-
sn
-glycero-3-phosphocholine (POVPC) for 2 hours before stimulation with 10% FCS for 24 hours. Cell proliferation and viability was measured for each condition using bromodeoxyuridine (BrdU) and ATP assays respectively. Incubation of POVPC caused a modest reduction in proliferation at the highest concentration (100μM, 58.16±11.3% of viable cells, n=6) while, PGPC dramatically reduced the number of viable cells in a concentration-dependent manner (25μM, 53.78±4.6%, n=6). Pretreatment of SMCs with an AMPK activator, A769662 for 45 mins, enhanced cell death to 10μM of PGPC (79.32±9.6% vs 102.96±5.8% for lipid alone, n=4). Pre-incubation with AMPK inhibitor, compound C for 30 mins, abolished the enhanced cell death at this concentration (88.54±14.9%, n=3). Similarly, activation of AMPK prior to 25μM POVPC treatment caused substantially more cell death than with lipid alone (48.01±13.24% vs 82.89±6.0% respectively, n=3). In contrast to PGPC, pretreatment with compound C had no effect on proliferation (25μM, 49.76±9.3%, n=3). Phosphorylated acetyl-CoA carboxylase, a downstream target of AMPK, showed a significant increase in expression after incubation of A76662 while no change after compound C was observed, analyzed by immunoblotting (4.10±0.95 vs 0.68±0.38, n=3). This novel study reveals that the activation of AMPK enhanced cytotoxicity of oxidized phospholipids, with PGPC and POVPC apparently operating through separate pathways. This contrasts with the protective effect usually observed for AMPK and may be critical in restenosis.
Abstract 185: The Impact of Cyclic Strain on Focal Adhesion Signaling and miRNA Profile in Vascular Smooth Muscle Cells
Authors: Mac Donnell Brian F
Published: March 2022

Vascular smooth muscle cells (VSCM) are major contributors to the regulation of vascular tone through their contractile flexibility. Along with other stimulus, they are exposed to mechanical cyclic strain. This is induced by changes in arterial blood pressure waveforms, generated from the left ventricle. It is thought such stimulus can be a major regulator of phenotypic modulation in smooth muscle cells. These altered states of VSMC fate can have a direct influence on cell function, including proliferation, apoptosis, migration and adhesion. This is considered to play a critical role in the pathogenesis of cardiovascular disease.

We have shown that cyclic strain, an important physiological stimulus, has a compounding influence on smooth muscle cell function via mechanotransduction. Increased strain has shown to increase SMC adhesion, proliferation and migration.

We hypothesise that the effect of mechanical cyclic strain is transduced through the cell via integrin rich focal adhesions, which can alter the miRNA signature of vascular smooth muscle cells and ultimately play a pivotal role in cell function. The Focal Adhesion Kinase (FAK) protein and its endogenous negative regulator FAK Related Non Kinase (FRNK) are known to play a commanding role in integrin signaling. We propose that down regulation of FAK by either siRNA technology or over expression of FRNK will negate the effect of pathological mechanical strain on cell function. We also predict regulation of expression on individual miRNA, through use of Pre-Mir and Anti-Mir technology could yield a similar effect.

In the current study we exposed primary derived human aortic smooth muscle cells, in an
in vitro
model, to cyclic strain to assess its influence on focal adhesions, integrin and miRNA expression. We have concluded thus far that increased cyclic strain shows increased cell migration, adhesion and proliferation. It also alters the miRNA signature as we have shown with a panel of mechano-sensitive miRNA. Coinciding with this we have also demonstrated the impact on cellular function from knockdown of miRNA biogenesis molecules such as Drosha, Dicer, Exportin 5 and Argonaute2 through use of siRNA.
Abstract 186: Effect of Direct Thrombin Inhibition on Vascular Function and Aortic Valve Function in Hypercholesterolemic Mice
Authors: Mehra Kashish , Hagler Michael A , Zhang Bin , Oehler Elise A , Van Ryn Joanne , Miller Jordan D
Published: March 2022

Thrombin plays a major role in coagulation cascade and participates in regulation of vascular function through its effects on protease-activated receptors (PARs). The effect of thrombin inhibition on vascular and aortic valve function during prolonged hypercholesterolemia, however, remains unknown. In the present study, we tested the hypothesis that direct thrombin inhibition (DTI, dabigatran etexilate) slows progression of endothelial and aortic valve dysfunction in ldlr-deficient, apolipoprotein B100-only (LA) mice. Carotid arteries from LA mice were used for studies of endothelial function in isolated organ baths, and aortic valve function was evaluated using echocardiography. In placebo-treated mice, maximum relaxation to acetylcholine was reduced following 6 and 9 months of hypercholesterolemia (maximum relaxation to acetylcholine (MR
ACH
) =75%± 3.7, and 67%± 3.6, respectively), but not affected by DTI (75%± 2.5, and 62%± 3.2, respectively). Relaxation to exogenous NO donors was also unaffected by DTI. Aortic valve function progressively deteriorated at 3, 6, and 9 months in placebo-treated mice (2.1 m/s± 0.04, 2.5 m/s± 0.1, and 2.9m/s ± 0.1, respectively), and was not affected by DTI (2.0m/s± 0.1, 2.5m/s± 0.1 and 3.0m/s ± 0.4, respectively). While PAR receptor expression was detected in aorta and aortic valve tissue, expression of SPHK1 (an indicator of PAR1 activity) was not altered by DTI. Expression of CTGF in aortic valves, however, tended to be reduced by DTI. In conclusion, our data suggest that DTI does not prevent vasomotor or aortic valve dysfunction in mice with prolonged hypercholesterolemia, but may attenuate pro-fibrotic signaling in aortic valves.
Abstract 190: Echium and Borage Oil Are as Atheroprotective as Fish Oil in Mice
Authors: Shewale Swapnil V , Boudyguina Elena , Hutchins Patrick M , Murphy Robert C , Parks John S
Published: March 2022

Background:
Diets enriched in long chain (>18 carbon) polyunsaturated fatty acids (PUFAs), such as 20:5 n-3 and 22:6 n-3 in fish oil (FO), are atheroprotective. However, most dietary fats consist of 18 carbon PUFAs that are poorly converted to longer chain PUFAs by the rate limiting enzyme, delta-6 desaturase (D6D).

Objective:
We investigated whether botanical oils enriched in 18 carbon fatty acids beyond the D6D step in the n-6 (borage oil, BO, 18:3 n-6 enriched) and n-3 (Echium oil, EO,18:4 n-3 enriched) pathway of elongation-desaturation are as atheroprotective as FO and the extent to which these botanical oils would affect oxidized CE species in the aorta.

Methods:
Female low density lipoprotein knockout (LDLr-/-) mice were fed atherogenic diets containing 0.2% cholesterol and 10% calories as palm oil (PO) with an additional 10% of calories as PO, EO, FO, or BO for 16 weeks. Periodic blood samples were taken for lipid and lipoprotein measurements and aortas were harvested after 16 weeks to measure cholesterol content and oxidized CE species.

Results:
Compared to the PO group, mice fed BO, EO and FO had significantly lower plasma total and very low density lipoprotein cholesterol concentrations. EO and FO groups also had lower plasma triglyceride concentrations compare to PO and BO fed mice. Compared to the PO group, mice fed BO and EO had significantly reduced aortic CE and free cholesterol, comparable to the FO group. Oxidized CE species were not detected in aortas of any diet group.

Conclusion:
Botanical oils enriched in 18 carbon n-6 or n-3 fatty acids beyond D6D in the elongation-desaturation pathway are as atheroprotective as FO in LDLr
-/-
mice. Furthermore, mouse aorta oxidized CE species were undetectable; suggesting this oxidized lipid species has a minimal role in mouse atherosclerosis progression.
Abstract 194: Determinants of Platelet Activation in Chronic Kidney Disease
Authors: Vazzana Natale , Liani Mario , Giacci Luciano , Del Rosso Goffredo , Salvati Filippo , Lattanzio Stefano , Liani Rossella , Davì Giovanni
Published: March 2022

Background.
Mechanisms of accelerated atherothrombosis in patients with chronic kidney disease (CKD) are only partly characterized. The aims of this study were to evaluate the extent of thromboxane (TX)-dependent platelet activation in patients with CKD, and to characterize the determinants of altered TX biosynthesis in this setting, with particular reference to enhanced lipid peroxidation and the hyperactivity of the ligand-RAGE axis.

Methods.
A cross sectional comparison between urinary 8-iso-PGF
2
α
and 11-dehydro-TXB
2
,
in vivo
markers of oxidative stress and platelet activation, respectively, and plasma endogenous secretory (es)RAGE has been performed in 95 patients with CKD and 20 healthy controls.

Results.
Urinary 8-iso-PGF
2
α
and 11-dehydro-TXB
2
were significantly higher in CKD patients as compared to healthy controls (P<0.0001). A significant direct correlation has been found between urinary 8-iso-PGF
2
α
and 11-dehydro-TXB
2
(Rho = 0.47, P < 0.0001). In CKD patients, urinary 11-dehydro-TXB
2
was inversely related with estimated glomerular filtration rate (eGFR) (Rho=-0.35, P=0.001) and hemoglobin levels (Rho = -0.53, P < 0.0001). In addition, plasma esRAGE levels were inversely related with eGFR (Rho=-0.30, P=0.006). On multivariate analysis, urinary 8-iso-PGF
2
α
and hemoglobin levels were the only independent predictors of urinary 11-dehydro-TXB
2
levels.

Discussion
. This study provides biochemical evidence of persistent platelet activation in patients with CKD. This condition occurs early in the natural history of the disease and is related to kidney function and oxidative stress. Moreover, we found an independent inverse relationship between hemoglobin levels and thromboxane-dependent platelet activation. This finding may provide mechanicistic link between CKD-related anemia and increased cardiovascular risk.
Abstract 197: MARCKS Signaling Modulates Vascular Smooth Muscle Cell Proliferation
Authors: Monahan Thomas S , Dong Tuo , Yu Dan , Bafford Richard A , Strickland Dudley K , Sarkar Rajabrata
Published: March 2022

Background:
The myrisolated alanine rich C-kinase substrate (MARCKS) is over expressed in animal models of intimal hyperplasia (IH) and in several human neoplasms. Further it has been demonstrated that MARCKS knock-down in vivo arrests vascular smooth muscle cell (VSMC) proliferation. We hypothesize that MARCKS arrests VSMC proliferation through a p27 dependent mechanism.

Materials and Methods:
Human coronary artery VSMC (Lonza) were grown in culture. Cell proliferation was determined by the AlamarBlue assay. Protein expression was determined by Western Blot analysis. MARCKS signaling was stimulated by treatment with the phorbol ester PMA, and MARCKS signaling was inhibited by either siRNA mediated knock-down or with one of two peptides that act as selective competitive antagonists.

Results:
Stimulation of VSMC in culture with PMA for 30 minutes resulted in and increased fraction of phosphor-MARCKS and complete knock-down of p27 protein. Treatment of VSMC in culture with siRNA resulted in arrest of cell proliferation, an increase in p27 expression with no change in p21 expression. Similarly, the peptide designed to specifically inhibit the functions of the N-terminal domain also resulted in similar effects in proliferation, p27, and p21 expression. The same results were noted when a separate peptide designed to inhibit the functions of the C-terminus domain was used to treat VSMC in culture.

Conclusion:
MARCKS signaling results in decreased p27 expression, which possibly explains proliferative VSMC phenotype observed in animal models of IH. Blockade of MARCKS signaling by siRNA mediated knock-down results in decreased cell proliferation and increased p27 protein expression. Antagonists to either the N- and C-terminus domains yield similar results in proliferation and p27 expression. VSMC proliferation requires both functional domains of MARCKS.