bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 441: Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) is Elevated in Acute Coronary Syndromes and Resides Predominantly in LDL
Authors: Jabor Bashar , Choi Hong , Ruel Isabelle , Hafiane Anouar , Genest Jacques
Published: March 2022

Lipoprotein associated phospholipase A2 (Lp-PLA2) may play a role in the formation of vulnerable atherosclerotic plaques and plaque rupture. Its plasma distribution and mass in subjects with acute coronary syndromes (ACS) are not characterized.

The objective of this study was to examine plasma levels and distribution of Lp-PLA2 in patient with ACS.

We compared plasma levels of LP-PLA2 in 23 patients within 48 hours of presentation of an ACS (visit 1) and 12 weeks after (visit 2), in 24 patients with chronic, stable coronary artery disease (sCAD) and in normal healthy controls. The distribution of Lp-PLA2 was determined by ultracentrifugation (UTC), high-performance liquid chromatography (HPLC), and polyethylene glycol (PEG) precipitation of plasma lipoproteins and immunoblotting analysis. Lp-PLA2 mass was determined by ELISA

The ACS patients (18 males/5 females, mean age 57±9.2) had hsCRP levels of 32.6±60.4 mg/L (ACS visit1) vs. 1.9±1.3 mg/L (ACS visit2), reflecting systemic inflammation. Stable CAD hsCRP 1.3±1.3 mg/L and controls 0.6±0.2 mg/L did not differ significantly. HDL cholesterol levels were 1.02±0.24 mmol/L for ACS visit 1, 1.00±0.3 mmol/L for ACS visit2, and 1.11±0.29 mmol/L for stable CAD. Plasma Lp-PLA2 levels were significantly higher in ACS visit1 subjects than in the ACS visit2 (144.3±60.6 vs. 89.5±40.0 ng/mL, p<0.0001). Interestingly, sCAD patients had slightly higher Lp-PLA2 levels that ACS visit 2 (p=0.003).

The distribution of Lp-PLA2 differed according to the technique used. We found that only approximately 45% of Lp-PLA2 mass remained in the lipoprotein fraction (d<1.21 g/L) after UTC, with the majority (43%) in the LDL fraction and only <3% in the HDL fraction. The remainder (55% was found in the d>1.21 g/L, suggesting that Lp-PLA2 is only loosely bound to lipoproteins, and the majority resides in LDL.

Here, we show that subjects with an ACS have markedly increased Lp-PLA2 levels acutely and that these levels fall within 12 weeks. Surprisingly, Lp-PLA2 is only loosely bound to lipoproteins and may be displaced in inflammatory conditions. The increase in Lp-PLA2 in ACS may contribute to the generation of oxidized fatty acyls and lysophopsphlipids and potentially compound the local inflammatory reaction.
Abstract 447: Treatment of Omega-3 Nutritional Deficiency Improves Cardiovascular Disease Risk Factors: Results of the Randomized, Double-Blind, Placebo-Controlled VASCAZEN-REVEAL Trial
Authors: Shaikh Nisar A , Ali Abbas , Yantha Jason , Shaikh Sabah , Jackowski George
Published: March 2022

Introduction

Treatments with Omega-3 (OM3) formulations have been suggested to have cardio-protective effects. A strong association of high plasma OM3 levels has been correlated with reduced risk of sudden death and “risk quartiles” based upon OM3 levels, have been previously described. However, systematic studies describing the extent of OM3 deficiency, it’s correction by intervention, and improvement in CVD risk factors are lacking. Our study is the first to determine dietary levels of OM3 in plasma and in red blood cells using Omega-Score and Omega-3 Index diagnostics, improvement after treatment with a proprietary “>90%-pure OM3 preparation with a 6:1 EPA (eicosapentaenoic acid): DHA (docosapentaenoic acid) ratio” (6:1-OM3), and the concomitant beneficial effects on CVD risk factors in patients with high triglycerides (TG 200-500mg/dL).

Hypothesis

CVD patients are nutritionally deficient in OM3 fatty acids, and through treatment with 6:1-OM3, such deficiency can be corrected, improving CVD risk factors.

Methods

A double blind, placebo-controlled study comprised of 110 subjects randomized and stratified by baseline TG levels (A: 90-199 mg/dL, and B: 200-500mg/dL). This report includes placebo (corn oil, n=54) and 6:1-OM3 treatment (> 3g of EPA + DHA/day; n=56) groups at baseline and after 8 weeks of treatment. The primary endpoints were the change in the Omega-Score (OS) and Omega-3-Index (OI), with secondary endpoints including the change in serum TG, lipoprotein cholesterol (VLDL, LDL, HDL, ApoB, and subfractions), and hsCRP.

Results

89% of screened CVD subjects (N=655) were nutritionally deficient in Omega-3. In group B subjects, a significant (p<0.0001) increase in OS (121%) and OI (112%) was observed in treated subjects with a TG reduction of 48% (p=0.0005), VLDL-C reduction of 30% (p=0.0023) and HDL-C increase of 9% (p=0.0069) without significantly affecting LDL-C, ApoB or hsCRP levels.

Conclusions

This study confirms that omega-3 deficiency is prevalent with CVD, and that such a deficiency can be corrected with 6:1-OM3 resulting in a concomitant and significant placebo-corrected reduction in TG and VLDL, and increase in HDL-C in patients with high TG (200-500mg/dL), without adversely affecting LDL-C.
Abstract 45: Critical Residues of the Hemoglobin a / Endothelial Nitric Oxide Synthase Macromolecular Complex Within Endothelial Cells Can Regulate Arterial Reactivity and Blood Pressure
Authors: Straub Adam C , Mutchler Stephanie M , Isakson Brant E
Published: March 2022

Arterial vascular reactivity is a fundamental process to the regulation of blood pressure, blood flow and oxygen delivery and is governed in part by endothelial nitric oxide (NO) synthase (eNOS). We recently demonstrated that monomeric hemoglobin α (Hb α) is 1) expressed in arterial endothelial cells, 2) enriched at the myoendothelial junction, 3) forms a complex with eNOS and 4) regulates the effect of eNOS signaling during vascular reactivity. Based on these results, we hypothesized that a macromolecular complex may form between Hb α and eNOS to regulate NO signaling. In silico modeling of potential Hb α and eNOS protein-protein interaction sites identified a discreet pocket and sequence where Hb α and eNOS may interface. Therefore, we created a TAT-linked peptide identical to the Hb α sequence (Hb α-X) along with a scrambled control (Hb α-scr). Using recombinant eNOS and Hb α protein, we demonstrate by coimmunoprecipitation a loss of protein-protein interaction in the presence of the Hb α-X peptide but not the Hb α-scr. Consistent with these results, the Hb α-X peptide incubated with thoracodorsal arteries exhibited disruption of the Hb α and eNOS complex measured by coimmunoprecpitation and by a proximity ligation technique. Next, we performed vascular reactivity studies, which resulted in significantly altered responses to both phenylephrine and acetylcholine with the Hb α-X peptide, which were reversed with a NOS inhibitor. To test the in vivo effect of these peptides on blood pressure, we administered the peptides to mice via intraperitoneal injection and performed real-time measurements using radio telemetry. Mice injected with the Hb α-X peptide showed a significant decrease in blood pressure (MAP= -10 mmHg). Collectively, these results identify a novel Hb α sequence that is critical for Hb α/eNOS interaction, control of arterial vascular reactivity and regulation of blood pressure.
Abstract 450: Effects of Multiple Infusions of Recombinant LCAT on Plasma and Tissue Lipids and Expression of Genes Involved in Atherogenesis
Authors: Vaisman Boris , Rousset Xavier , Auerbach Bruce J , Krause Brian R , Homan Reyn , Freeman Lita , Ossoli Alice , Sampson Maureen L , Remaley Alan
Published: March 2022

BACKGROUND

LCAT, an enzyme that converts small, discoidal HDL to spherical HDL particles by esterifying free cholesterol, plays a key role in reverse cholesterol transport (RCT). LCAT replacement therapy holds promise for treatment of familial LCAT deficiency, as well as a possible acute treatment in patients with Acute Coronary Syndrome.

HYPOTHESIS

Injecting recombinant LCAT into LCAT-deficient mice expressing human apoA-I will increase plasma HDL-C levels, improve reverse cholesterol transport, and alter expression of genes involved in atherosclerosis.

METHODS

We injected recombinant human LCAT or vehicle intravenously daily for 4 days into LCAT-KO x human apoA-I transgenic mice (n=4-6 in each group). The dose of rLCAT was approximately 10-fold larger in terms of mass than the endogenous levels of mouse LCAT. Lipids in plasma and tissues, as well as expression of 84 cholesterol metabolism genes in control and experimental groups were measured.

RESULTS

Total cholesterol (TC) in plasma increased from 80 (vehicle) to 473 mg/dl (LCAT) (
P
<0.002) after rLCAT treatment. Cholesteryl esters (CE) increased from 8 (vehicle) to 339 mg/dl (LCAT). Lipids were localized mostly in the HDL fraction. The accumulation of lipids in plasma was accompanied by decreased lipid content in peripheral tissues.TC in aorta, heart, kidney and spleen decreased, by 15%; 27%; 13% and 27%, respectively (
P
<0.05). In kidney, lung and spleen, CE decreased from 17; 18 and 28 nmol/mg dry weight, respectively, to being below detection. LCAT injections did not significantly alter liver lipid content, but did significantly increase expression of cholesterol synthesis genes (Dhcr7, Hmgcr, Mdv, Mvk and Tm7sf2) and decreased expression of the cholesterol efflux gene Abcg1.To further investigate the potential of interaction between apoA-I and LCAT, we also studied apoA-I-Tg x LCAT-Tg double transgenic mice. Their TC in plasma reached more than 1300 mg/mL, with similar gene expression changes observed in hapoA-I transgenic mice but more pronounced.

CONCLUSIONS

The results show the feasibility of using recombinant LCAT as a therapy for LCAT deficiency and for other diseases associated with accumulation of cholesterol in tissues.
Abstract 453: Is There a Relationship Between Platelet Count and Atherosclerosis Determined by Calcium Score in Patient With Acute Chest Pain?
Authors: Kassi Mahwash , Chaikriangkrai Kongkiat , Khan Zahoor , Khaleel bala Sayf , Cordero Andrea , Chang Su Min
Published: March 2022

Background

Platelet interaction and the clotting cascade are known to play an important role in the process of atherosclerosisHowever, the relationship between platelet count and calcium scores is not well known.

Methods

This is a sub-group analysis of 974 consecutive patients who presented to our institution for chest pain without evidence of myocardial infarction. Calcium scores were determined in these patients using cardiac CT scans. The platelet count was divided in quartiles. Pearson’s correlation was used to test association between platelet count and calcium scores.

Results

The predominant gender was female (60%) and the mean age was 57. Sixty percent of the population was Caucasian. The mean Framingham score was 8.16 ± 8.15. There was no difference seen in the prevalence of low calcium scores (100) in different platelet groups when divided in quartiles. Similarly, there was no difference in mean calcium scores in different groups.

Conclusion

The serum platelet count does not correlate with calcium scores.
Abstract 458: Alternatively-Spliced Extra Domain A of Fibronectin Exacerbates Atherosclerosis via a Toll-Like Receptor 4- Dependent Mechanism
Authors: Gandhi Chintan , Ahmad Ajmal , Chauhan Anil K
Published: March 2022

Rationale

Fibronectin containing the alternatively-spliced extra domain A (EDA
+
-FN) is absent in healthy arteries, but expressed in the atherosclerotic arteries of humans and mice. The mechanistic role of EDA
+
-FN in the pathophysiology of atherosclerosis remains unclear. Recent studies by us and others suggest that EDA
+
-FN is a ligand for toll-like receptor 4 (TLR4).

Hypothesis

EDA
+
-FN exacerbates atherosclerosis via TLR4 pathway in ApoE
-/-
mice.

Model and Methods

We generated EDA
+/+
/ApoE
-/-
(constitutively over express EDA
+
-FN) and EDA
-/-
/ApoE
-/-
mice (EDA-deleted allele of FN and express FN lacking EDA). ApoE
-/-
mice was used as control. To elucidate the role of TLR4 in EDA
+
-FN-mediated atherosclerosis, we generated EDA
+/+
/TLR4
-/-
/ApoE
-/-
and EDA
-/-
/TLR4
-/-
/ApoE
-/-
mice. Male mice (n=12-14/group) were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and
en face
lesion area measured by morphometry, and in the cross section area of the aortic sinus using the Verhoeff-Van Gieson stain. Macrophage infiltration was quantified by immunohistochemistry.

Results

We report that atherosclerotic plaque formation in the aortae and aortic sinus of EDA
+/+
/ApoE
-/-
mice was increased by 2-fold, concomitant with significant increased macrophage infiltration (% of total plaque area) (
P
<0.001 vs.ApoE
-/-
mice, ANOVA). Deletion of the EDA exon of FN gene in ApoE
-/-
mice significantly reduced atherosclerotic plaque formation (aorta and aortic sinus) and macrophage infiltration (
P
<0.01 vs. ApoE
-/-
mice, ANOVA). Total cholesterol and triglycerides levels were similar among groups. TLR4 deficiency in EDA
+/+
/ApoE
-/-
mice significantly reduced atherosclerotic plaque formation in the aorta and aortic sinus, concomitant with decreased macrophage infiltration in aortic sinus (
P
<0.05 vs. EDA
+/+
/ApoE
-/-
mice, Two way ANOVA), whereas TLR4 deficiency in EDA
-/-
/ApoE
-/-
mice had no effect compared to EDA
-/-
/ApoE
-/-
mice.

Conclusion

These findings reveal a mechanistic role of endogenous EDA
+
-FN in modulating atherosclerosis via a TLR4-dependent pathway.
Abstract 460: The Molecular Mechanism of Regulation of Macrophages Pinocytosis of Lipoproteins by Akt3
Authors: Ding Liang , Podrez Eugene
Published: March 2022

Akt, also known as protein kinase B (PKB), plays an important role in the regulation of numerous cellular processes, including cell growth, glucose metabolism, differentiation, proliferation, migration and apoptosis. We have recently demonstrated that deletion of Akt3 in macrophages promotes foam cell formation by increasing the uptake of native and modified LDL (Low-density lipoprotein) and elevating the level of ACAT-1, an enzyme esterifying excess cholesterol and promoting its storage in cytoplasmic lipid droplets. We ruled out increased scavenger receptor-dependent uptake and receptor-dependent endocytosis of lipoproteins as the mechanism of increased lipoprotein uptake, and demonstrated in vitro enhanced receptor-independent pinocytosis in Akt3-/- macrophages. The aim of this study was to investigate the mechanism of regulation of macrophage pinocytosis by Akt3. Actin assembly is the pivotal process in pinocytosis of lipoproteins. We observed increased actin assembly in Akt3-/- macrophages assessed by phaloidin staining, suggesting that actin assembly can be involved in the elevated pinocytosis in Akt3-/- macrophages. The increase in pinocytosis by Akt3-/- macrophages was reduced in the presence of actin assembly inhibitor, indicating that the enhanced pinocytosis in Akt3-/- macrophages is actin assembly dependent. Colocalization of actin filaments with Akt3 in wild-type murine macrophages using a confocal fluorescence microscopy as well as coimmunoprecipitation of β-actin with Akt3 in transfected HEK293 cells revealed a direct interaction between Akt3 and β-actin. We ruled out the possibility that Akt3 regulates macrophage pinocytosis via major downstream molecules of the Akt signaling pathway (mTOR, GSK3 and NFκB) by using specific pharmacological inhibitors. However, we observed that increased pinocytosis in Akt3-/- macrophages is Src kinases dependent. These results suggest that Akt3 may inhibit actin assembly and subsequent pinocytosis of LDL by direct interaction with β-actin. Taken together, our studies describe a novel mechanism of atheroprotective role of Akt3.
Abstract 467: Nitric Oxide-Mediated Regulation of the Adiponectin-Adiponectin Receptor Pathway Following Arterial Injury
Authors: Koo Nathaniel C , Havelka George E , Martinez Janet , Flynn Megan , Kibbe Melina R
Published: March 2022

Objectives

Nitric oxide (NO) has been shown to limit the development of neointimal hyperplasia; however, little is known about its effects on the adventitia. Adiponectin is a molecule that originates from periadventitial adipose tissue and has been shown to negatively regulate neointimal growth. Thus, we hypothesize that removal of periadventitial fat from the arterial wall will negatively impact the ability of NO to inhibit the development of neointimal hyperplasia following injury.

Methods

The carotid artery injury model was performed on 11-week-old Sprague Dawley rats. Animal models underwent either removal of all periadventitial fat from the artery at the time of surgery (absent) or no disturbance of the periadventitial fat (present). Treatment groups included control, injury, and injury + NO (PROLI/NO, 10mg). Arteries were harvested at 2 weeks. Morphometric analysis was performed using ImageJ software. Immunofluorescent staining was performed for the adiponectin receptor (AR) and assessed using blinded grading (scale 0-4).

Results

Morphometric analysis revealed a 29% increase in neointima development in the absent vs. present group (p<0.001). With the application of NO, there was a greater decrease in intima area in the absent than the present group (93% vs. 77%, p<0.001). The results were similar for media area (50% vs. 37%, p<0.001) and the intima/media area ratio (86% vs. 65%, p<0.001). While AR staining showed no significant difference in the media or adventitia with the application of NO in the present group, AR staining was significantly increased in both the media (57%, p=0.009) and adventitia (37%, p<0.05) with the application of NO in the absent group.

Conclusions

Removal of the source of periadventitial adiponectin resulted in more neointimal hyperplasia following arterial injury, but a paradoxical NO-mediated increase in AR levels and corresponding responsiveness to NO therapy. These data suggest that AR expression is upregulated in the absence of adiponectin by NO. Further studies that identify the mechanism by which NO may regulate adiponectin may provide a novel therapeutic target to prevent neointimal hyperplasia.
Abstract 47: Role of Indoleamine 2,3-dioxygenase in Atherosclerosis Development
Authors: Taleb Soraya , Ponnuswamy Padma , Simon Tabasome , Tedgui Alain , Mallat Ziad
Published: March 2022

Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme that catalizes the degradation of tryptophan along the kynurenine pathway, is seen as a major mediator of immune tolerance. Our purpose is to study the role of IDO in an inflammatory disease which is atherosclerosis. We showed that surprisingly the total IDO deletion in

LDLr
-/-

mice (

LDLr
-/-
IDO
-/-

) or in mice reconstituted with

IDO
-/-

bone marrow showed a significant reduction in atherosclerosis (p<0.05). Then, we examined the inflammatory profile of the cells known to express IDO such as macrophages and plasmacytoid dendritic cells (pDC). Interestingly,

IDO
-/-

macrophages or Flt3 ligand-induced bone marrow-derived dendritic cells (mostly plasmacytoid.DC) showed a significant increase of IL-10 expression compared with controls (p<0.05).

To examine the role of IL-10 in IDO-mediated effect, we made double knockout- mice
IDO

-/-
IL-10
-/-

. Importantly, the resistance of IDO-deficient mice to enhanced atherosclerosis and immune activation is broken in IDO
-/-
IL-10
-/-
mice, which then show exaggerated immune responses and display signs of autoimmunity such as splenomegaly and significant increase of ssDNA and dsDNA auto-antibodies. We also showed that kynurenic acid, a kynurenine-derived metabolite, is responsible for reduced IL-10 production through inhibition of Erk1/2 phosphorylation and then increased atherosclerosis development (p<0.05). Our results reveal a previously unsuspected Janus-faced IDO that actively represses IL-10 while promoting other pathways of immune tolerance, thereby introducing a higher degree of complexity in immune regulation.
Abstract 472: Fractional Flow Reserve (FFR): FAR FROM REALITY
Authors: Patel Chintan , Ahmed Sameer , Morsy Mohamed
Published: March 2022

Fractional Flow Reserve (FFR) is the ratio of maximal blood flow in a stenotic artery to normal maximal flow. It is the “gold standard” technique used in diagnostic angiography to determine whether percutaneous intervention is needed in a moderate coronary stenosis (estimated 50-70% lesion). An FFR less than 0.8 may be falsely reassuring in a patient with unstable plaque who may be actively forming and dissolving clot.

This is a case of a 36 year old gentleman, with no significant past medical history, who presented to another local hospital with complaints of episodic chest pain that was located in the sub-sternal region and radiated to his left upper extremity. He was found to be mildly hypertensive, with elevated cardiac markers and with no EKG findings. On cardiac catheterization the patient was found to have a 50% lesion of left anterior descending artery and an FFR measurement of 0.93-0.91, therefore the lesion was not stented. The patient was subsequently discharged home. Two months later the patient presented to the hospital with crushing chest pain, and found to be having a ST elevation myocardial infarction. On repeat cardiac catheterization, the patient was found to have a 99% lesion of his left anterior descending coronary artery requiring placement of a drug eluting stent.

Cardiac catheterization is a life saving diagnostic and interventional modality for many patients with coronary artery disease. Fractional Flow Reserve is a new diagnostic tool used to guide management of coronary lesions with intermediate stenosis. It does not, however, take into account the pathophysiology of the lesion itself, for example a stable versus an unstable plaque. As in this case, a non flow-limiting lesion can still be unstable and plaque rupture can create a life-threatening myocardial event. With the development of intravascular ultrasound and optical computed tomography, the ‘gold standard’ seal of FFR will require reevaluation in regards to diagnostic modalities.
Abstract 473: Fluid Shear Stress Specifically Exacerbates Chlamydia pneumoniae-mediated Pro-inflammatory Responses in Monocytes
Authors: Evani Shankar J , Dallo Shatha F , Ramasubramanian Anand K
Published: March 2022

Background and Significance

The precise role of Chlamydia pneumoniae infection in various stages of the atherosclerosis is not well understood. During the transit from lung to the atherosclerotic sites, C. pneumoniae-infected monocytes travel through circulation and are subjected to shear stress due to blood flow. Elucidating the effects of these mechanical stimuli on infected monocytes is critical in our understanding of the link between C. pneumoniae infection and atherosclerosis.

Objectives

We hypothesized that fluid shear stress alters the inflammatory response of C. pneumoniae-infected monocytes. Using an in vitro model of blood flow, we determined the effect of physiological levels of shear stress on monocyte responses pertinent to atherosclerosis including cytokine secretion, adhesion molecule expression, intracellular signaling, and endothelial adhesion.

Methods

Primary human monocytes and THP-1 cells were infected with C. pneumoniae at an MOI-2 for 2 h at 35 °C with intermittent rocking, and cultured for 36 h. The infected cells were then subjected to a shear stress of 7.5 dyn/cm2 for 1 h, and the supernatants were analyzed for a panel of 17 cytokines, and the cells were analyzed for signaling proteins, surface receptors, and endothelial adhesion. Uninfected cells and static conditions were used as controls, and a P<0.05 (Student’s t-test) was considered significant.

Results

We observed that shear stress on infected cells resulted in a caspase-independent upregulation of pro-inflammatory IL-1β (3-fold), a modest increase in MIP-1α and MIP-1β, a decrease in anti-inflammatory IL-10, and no change in other cytokines including TNFα and IL-12. These changes were manifested as an increase in the adhesion of monocytes to endothelium activated with the infected supernatants. We also observed differences in the chemotaxis of monocytes exposed to sheared vs. static supernatants.

Conclusions

We observe that physiological shear stress drives Chlamydia infected monocytes towards a pro-inflammatory state in circulation suggesting a critical synergy between mechanical and chemical factors in atherogenesis.
Abstract 480: Serum Amyloid A Acts Through TGF-ß to Increase Atherosclerosis in ApoE Deficient Mice
Authors: Thompson Joel C , Yoder Meghan H , Wilson Patricia , Brandewie Kyle , Webb Nancy , Tannock Lisa R
Published: March 2022

Serum amyloid A (SAA), along with high sensitivity C- Reactive Protein, is a clinical marker of atherosclerosis, the leading cause of death in westernized countries. SAA is modestly but chronically elevated in several diseases such as diabetes and obesity that confer greater risk to developing atherosclerosis. It was recently reported that elevated SAA is pro-atherogenic in a murine model, although the mechanisms remain unclear. As outlined in the response to retention hypothesis, proteoglycan mediated lipoprotein retention is thought to be a key step in the initiation of atherosclerosis. We previously demonstrated in vitro that vascular smooth muscle cells treated with SAA had increased vascular biglycan synthesis with increased LDL binding affinity. We further demonstrated that SAA stimulation of biglycan was mediated via induction of TGF-β. To evaluate the pro-atherogenic effects of SAA in vivo, ApoE-/- male mice were injected with an adenovirus carrying the gene for human SAA1 (adSAA), a null control (adNull) or saline and maintained on chow for 16 weeks. SAA was elevated immediately after injection of adSAA but returned to baseline by 10 days. Although the SAA elevation was short, mice injected with adSAA had increased vascular biglycan content and significantly increased atherosclerosis at the aortic sinus (p<0.05) and en face aorta (P<0.001) compared to controls. SAA, apoB and biglycan co-localized within atherosclerotic lesions. To determine if TGF-β was required for the pro-atherogenic effects of SAA ApoE deficient mice received either a TGF-β neutralizing (1D11) or control antibody (13C4) simultaneously with injection of adSAA or adnull. The administration of TGF-β neutralizing antibody 1D11 prevented SAA-stimulated atherosclerosis but the irrelevant antibody 13C4 had no effect. Thus, in apoE-/- mice, SAA accelerates the development of atherosclerosis in a TGF-β[[Unable to Display Character: ]]dependent manner.
Abstract 491: Protein Kinase D2 Mediates LPA-induced Tissue Factor Expression and Activity through the MAPK Pathway in Mouse Aortic Smooth Muscle Cells
Authors: Hao Feng , Zhang Fuqiang , Xu Kan , Xu Xuemin , Cui Mei-Zhen
Published: March 2022

Lysophosphatidic acid (LPA), a potent bioactive lipid in oxidized-LDL, modulates vascular cell functions in vitro and in vivo via regulating the expression of specific functional genes. We previously reported that LPA highly induces the expression of tissue factor (TF) in vascular smooth muscle cells. TF is the principal initiator of blood coagulation and is important for thrombosis. However, the intracellular signaling pathway leading to LPA induction of TF is not well understood. In the current study, we found that LPA markedly induced protein kinase D (PKD) activation in mouse primary aortic smooth muscle cells (MASMCs). PKD consists of three isoforms (PKD1, PKD2 and PKD3), which belong to a family of serine/threonine protein kinases. We observed that all three PKD isoforms were expressed in MASMCs. LPA-induced PKD phosphorylation is via a PKC-dependent pathway. Employing a small-interfering RNA approach to knock down the expression of the specific isoforms of PKD in MASMCs, we found that knockdown of PKD2 expression blocked LPA-induced TF expression and activity, indicating that PKD2 is the key intracellular mediator for the expression and surface activity of TF. The role of PKD2 was further confirmed by overexpression of the dominant-negative PKD2. We observed that overexpression of the dominant-negative isoform of PKD2 blocked LPA-induced TF expression. Our data also demonstrate that PKD2 mediated LPA-induced TF expression via the mitogen-activated protein kinase pathway. Using primary MASMCs isolated from LPA receptor knockout mice, our data revealed that LPA receptor 1 was responsible for LPA-induced TF expression. Therefore, these data provide the first evidence that PKD is a new component in LPA signaling in vascular SMCs, leading to gene expression, as evidenced by TF expression. Our data provide new insights into the mechanisms of coagulation and possible therapeutic targets for atherothrombosis.
Abstract 494: Rho-a Kinase Activation in Endothelial Cells is Induced by Cocaine or Plasma From Cocaine Consumers: Association With a Prothrombotic Phenotype and Effect of the Inhibitors Atorvastatin and Y-27632
Authors: Saez Claudia G , Pereira-Flores Karla D , Mendoza Camila A , Ocaranza María P , Jalil Jorge E , Massardo Teresa , Mezzano Diego , Pallavicini Julio , Ibañez Carlos , Veliz Jonathan , Ebensberger Roberto , Pereira Jaime
Published: March 2022

Cocaine consumption is associated with increased risk of cardio- and cerebrovascular diseases, which are the consequence of endothelial dysfunction (ED). We recently showed that cocaine use is related to increased ED markers including circulating endothelial cells, although the mechanisms governing ED in cocaine users are still being researched. Since the RhoA/Rho kinase (ROCK) pathway is involved in the pathogenesis of ED, our
Aim
was to study
in vitro
and
ex vivo
RhoA/Rho kinase activation and ED markers in relation to cocaine consumption.

Method

Plasma samples from active cocaine consumers who met DSM-IV criteria and age and gender-matched controls were tested. ROCK activity, von Willebrand factor (vWf), Metalloproteases (MMP) and platelet adhesion were evaluated in HUVEC supplemented with cocaine (10μM), 10% plasma from cocaine users (CP), normal plasma (NP) or vehicle, in the presence or absence of Y-27632 (10 μM) and atorvastatin (ATS:10μM).

Results

CP induced higher ROCK (p: 0.039) and MMP 2 activity (p<0.05) in HUVEC, released significantly elevated vWf (p0.001), but failed to stimulate MMP activity or vWf release. ATS and Y-27632 significantly reduced ROCK activity (p<0.01 and p<0.001) and platelet adhesion (p< 0.0001 and p: 0.0073) in CP treated cells. ATS also reduced MMP2 activity (p< 0.001) and vWf release (p: 0.004). In cocaine treated cells, ATS and Y-27632 downregulated ROCK activity (p: 0.039 and p: 0.091) and significantly reduced platelet adhesion (p: 0.0156 and p: 0.073).
Ex vivo
studies showed significantly higher ROCK (p 0.015), MMP-2 (p<0.05) and MMP-9 (p<0.01) activity in cocaine consumers than in controls.

Conclusions

Cocaine and cocaine consumer plasma increased ROCK activity in HUVEC, resembling the
ex vivo
findings. Only CP induced higher MMP activity and release of vWf resembling the findings in patients. The significant reduction of most of the in vitro effects by ATS and Y-27632 may provide a novel and potential therapeutic tool for the protection of the endothelium from deleterious stimuli such as cocaine consumption.
Abstract 50: Absence of L-selectin Affects the Distribution of B Cell Subsets and Local Immune Response in Apoe-/- Aortas
Authors: Gjurich Breanne N , Ley Klaus , Galkina Elena V
Published: March 2022

To date, a significant body of evidence implicates the immune system in atherosclerosis. Multiple cell subsets provide a fine tuning of pro-inflammatory and suppressive arms of the immunological response. Studies report that follicular B cells are pro-atherogenic whereas B1a cells play a protective role via T15/E06 antibody production; yet the other B cell subsets have to be explored in atherosclerosis. Thus far, little information is available on the mechanisms behind the regulation of cell subset specific homing during atherogenesis. L-selectin is a homing receptor modulating leukocyte tethering and rolling. To investigate the role of L-selectin in atherosclerosis, we generated 50 week old L-selectin deficient

Apoe
-/-

(

Sell
-/-
Apoe
-/-

) mice fed a chow diet. A L-selectin deficiency resulted in accelerated atherosclerosis that was accompanied by a 1.3-fold increase in the number of leukocytes within aortas of

Sell
-/-
Apoe
-/-

mice. In contrast to the elevated total leukocyte cellularity in aortas,

Sell
-/-
Apoe
-/-

aortas had a 2-fold reduction in B cell content. Importantly, an L-selectin deficiency significantly reduced the percentage and number of B1a and regulatory B (B
reg
) cells within

Sell
-/-
Apoe
-/-

compared to

Apoe
-/-

aortas and spleens. Despite reduced numbers of B1a cells we detected a 3.2-fold increase in the splenic mRNA expression of T15/E06 suggesting that B1a cells are hyperactive, but are still unable to reduce atherogenesis in

Sell
-/-
Apoe
-/-

mice. To further examine the effects of L-selectin deficiency on aortic wall inflammation, we analyzed lymphocyte activation and found elevated mRNA expression of B and T cell activation markers in

Sell
-/-
Apoe
-/-

aortas. In line with the increased leukocyte cellularity of the aorta, there was a 1.5-fold increase in aortic CD68
hi
macrophages. These data provide evidence for a functional role of L-selectin in atherosclerosis via the regulation of the content of aortic B1a and B
reg
cells and the modulation of the local immune response within the aorta. However, it also suggests the functions of B1a and B
reg
cells can be suppressed by additional factors contributing to accelerated atherosclerosis.
Abstract 501: Poly Herbal Formulation Prevents Foam Cell Formation Possibly Through Attenuation of CD36 Scavenger Receptor Signalling Cascade
Authors: Nimgulkar Chetan C , Anand Shankar B , Kumar Uday P , Ghosh Sudip , Kumar Dinesh B
Published: March 2022

Objective

CD36 signalling cascade promotes migration, spreading and entrapment of lipid laden foam cells within intimal lesions by oxidative stress, inflammation and oxidized LDL (oxLDL) by activation of actin polymerization, Focal Adhesion Kinase (FAK) and cement adhesion which also are indirectly activated by Reactive Oxygen Species (ROS). There is dearth of information about an antagonistic therapy for this CD36 cascading pathway to prevent atherosclerosis. Therefore, we hypothesized that, an antioxidant, antiinflammatory and antihyperlipidemic Poly Herbal Formulation (PHF) consisting of Allium sativum, Zingiber officinale, Curcuma longa, Terminalia arjuna and Cyprus rotundus prevents atherosclerosis.

Methods

Standardized (WHO and IP monographs) hydro-alcoholic extracts of the above said test materials were mixed in equal amounts to prepare PHF. After conforming the antioxidant, antiinflammatory and antihyperlipidemic activities, PHF was evaluated for foam cell formation and the protein expression of CD36, PPAR-γ and FAK in oxLDL treated mouse macrophage J774A.1 cells. Its inhibitory activity for ROS generation was evaluated. Antiatherosclerotic activity was studied in high cholesterol fed rabbit model.

Results

Significant accrual of lipid in oxLDL treated cells conform the foam cell formation where CD36, PPAR-γ and FAK protein levels had increased. PHF (20 to 80 μg/mL) prevented lipid accumulation in oxLDL treated cells from ~29% to ~38% where CD36, PPAR-γ and FAK protein levels were significantly decreased in concentration dependent manner up to 3, 2 and 2 folds, respectively. ROS generation was inhibited by PHF in concentration dependent manner.

PHF dose of 100 and 200mg/Kg, significantly reduced plasma cholesterol, ~23% and ~40% and plasma triglycerides ~87% and ~91%, respectively. Plasma TBARS were significantly reduced by PHF 100 and 200mg/Kg, 50% and ~66%, respectively. Aortic atheroma formation was significantly barred by PHF as observed in histopathological studies.

Conclusion

PHF prevents atherosclerosis by preventing foam cell formation possibly by reducing CD36, PPAR-γ and FAK proteins and ROS generation. However, detailed pharmacodynamic studies are needed to correlate these findings in a clinical set up.
Abstract 502: Arterial Calcification Increases Neointimal Proliferation Following Balloon Injury in Rat Carotid Arteries
Authors: Marshall Andre P , Wang Xue-lin , Paone Darien A , Lin Tonghui , Luo Weifeng , Guzman Raul J
Published: March 2022

Introduction

Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We sought to determine how calcification affected neointimal accumulation after balloon injury in the rat carotid.

Hypothesis

We propose that transformed SMCs in calcified arteries respond to balloon injury in an accelerated manner through a mechanism involving bone-related factors.

Methods

Arterial calcification was induced in male Sprague-Dawley rats weighing 350-400 grams by subcutaneous administration of vitamin D3. Control rats received vehicle. Both groups underwent left carotid balloon injury one week following injection, a time at which significant medial calcification has developed. Arteries were harvested twenty-one days following balloon injury. The degree of neointimal cell proliferation was next assessed by immunohistochemical staining for PCNA. Neointimal hyperplasia was measured by morphometric analysis using standard imaging analysis software.

Results

At 3 weeks after carotid balloon injury, mean cellular proliferation as measured by PCNA staining was higher in the intima of calcified than control arteries (63.8 ± 6.0 versus 40.5 ± 9.5 cells/hpf, P=ns). Calcified arteries from Vitamin D3-treated rats, however, had significantly more intimal hyperplasia than control uncalcified arteries (intima-to-media ratio = 1.374 ± 0.071 versus 0.758 ± 0.080, P=0.0021). Intimal and medial areas were also individually larger in calcified than control arteries.

Conclusions

Neointimal hyperplasia is accelerated in Vitamin D3-treated rats through a mechanism that partially involves increased SMC proliferation. Calcified vessels may respond differently to balloon angioplasty and this may explain the poorer results of endovascular interventions in patients with arterial calcification. Other factors including accelerated migration and increased matrix synthesis may also be involved. Treatments to inhibit restenosis in calcified arteries may differ from those that work in uncalcified vessels.
Abstract 506: The Effect of Human Recombinant DNase-1 Treatment on Skeletal Muscle Ischemia Reperfusion Injury
Authors: Albadawi Hassan , Oklu Rahmi , Malley Rita , Yoo Hyung-Jin , Austen William G , Watkins Michael T
Published: March 2022

Objective

Nucleosomes are known to exacerbate tissue injury, thus exogenous DNase-1 has been suggested as a novel therapeutic strategy to limit tissue injury in a stroke model. Our aim was to assess the effect of recombinant DNase-1 treatment on skeletal muscle fiber injury, tissue inflammation and levels of circulating nucleosomes in a mouse model of hind limb ischemia-reperfusion-injury (IR).

Methods

C57BL6 mice were subjected to IR (1.5hr Ischemia/24hr Reperfusion) or sham conditions. Mice were treated with either 50μg DNase-1 IP every 6 hours (DNase-1) or buffer (Control) at the same time intervals. Sham mice were anesthetized for 1.5 hours and allowed to recover for 24 hours. Muscles were harvested to quantify muscle fiber injury, DNAse-1 protein and calcium dependent endonuclease activity. Muscle markers of inflammation Myeloperoxidase (MPO), Keratinocyte chemoattractant (KC) and Plasma nucleosomes were quantified.

Results

Skeletal muscle fiber injury (numbers p<0.05) and endonuclease activity (numbers p<0.001) were greater in the DNase-1 group compared to control and sham. DNase-I localized predominantly to the extracellular matrix and severely injured muscle fibers. There was no significant difference in the plasma levels of nucleosomes or markers of inflammation (MPO or KC).

Conclusions

These novel findings suggest that in contrast to brain IR, hind limb IR increases DNAse-1 activity, and that exogenous treatment with DNase-1 exacerbates muscle injury independent of markers of inflammation. DNase-1 may be a therapeutic target that warrants further investigation.

Table to Be Inserted
Abstract 509: Inhibition of Patched-1 prevents Injury-induced Neointimal Hyperplasia
Authors: Redmond Eileen M , Hamm Katie , Cahill Paul A , Morrow David
Published: March 2022

Intimal remodeling occurs due to alterations in vascular smooth muscle cell (VSMC) proliferation and apoptosis and is a key event in the pathogenesis of vascular disease. Atherosclerosis, vascular rejection, venous graft restenosis and coronary intervention are each characterized by increased VSMC growth and intimal thickening.

Objectives

To determine the role of Ptc-1 in mediating pulsatile flow-induced changes in SMC growth and neointimal hyperplasia.

Methods and Results

HCASMC were exposed to “normal“ or pathological “low” pulsatile flow conditions for 24 h by utilizing a perfused transcapillary flow system. In vivo, pathological low flow conditions following carotid artery ligation increased medial SMC growth while increasing Ptc-1/Notch expression. Inhibition of Ptc-1 expression by cyclopamine attenuated low flow-induced increases in VEGF/Notch expression while concomitantly decreasing HCASMC growth to normal flow levels. Perivascular delivery of Ptc-1 siRNA by pluronic gel inhibited the low flow-induced increases in Ptc-1/Notch expression and subsequent increase in medial SMC growth. In addition, low pulsatile flow-induced increases in neointimal hyperplasia and subsequent decreased lumen volume were returned to sham-operated control levels following ptc-1 gene knockdown.

Conclusion

These results suggest that pathological low flow stimulates SMC growth and neointimal hyperplasia via Ptc-1 regulation of VEGF/Notch signaling.
Abstract 510: Morphometrics of Calcification in Carotid Endarterectomy Tissue
Authors: Han Richard I , Chen Iou , Brunner Gerd , Morrisett Joel D
Published: March 2022

Arterial calcification plays a significant role in the pathogenesis of carotid atherosclerotic plaques development. The aim of this study was to provide a detailed analysis of calcification morphology based on histological features in carotid endarterectomy (CEA) specimens. In particular, the emphasis is on calcium nodule size/shape and its relation to tissue and lumen morphologies. These studies will help elucidate the mechanisms of the arterial calcification process.

Representative frozen sections were cut from the common, bifurcation, internal and external segments obtained from 9 CEA samples. Histological stains were used to show the calcified particles, tissue compositions and lipids deposition. The digitized images of sections were warped when necessary to achieve closure of the vessel wall. Image analysis was applied to 2 samples to determine the morphologies of calcium particles (size, shape and perimeter), lumen (area and perimeter) and tissue (area and compositions) using ImageJ. Total non-collagen protein and collagen protein were semi-quantitatively analyzed in one sample.

Calcification occurred predominantly in the bifurcation and internal segments of the CEA, occupying up to 17% of the cross-sectional tissue area. Occassionally, calcification was found in the external branch. The lipids distribution is quite patchy. All calcified particles were contained in the intimal layer of the carotid wall. The lumen area was increased in the bifurcation segment, but it deviated from a patent circular shape. The amount of calcific particles was positively correlated with the tissue area (R2=0.8365, p=0.0015). As the calcium particle increase in size, the shape became more elongated. Collagen accounted for up to 5% of the total non-collagen protein.

Different stages of calcification progression occur at the same time, while the very small sized calcium particle grow in size in all directions, the medium sized particle coalescing with its neighbors to form larger particles. The calcification is accompanied by increase in tissue size and distortion of lumen. Presumably, the kinetics of calcification is determined by the composition of the extracellular matrix and the interstitial cells may play a role in the process.
Abstract 513: Role of Membrane-Type 1 Matrix Metalloproteinase in the Pathogenesis of Thoracic Aortic Aneurysms
Authors: Ward Alison F , Mignatti Paolo , Grossi Eugene A , Ursomanno Patricia , Maddalo Scott , DeAnda Abe
Published: March 2022

Background

The histopathologic hallmark of thoracic aortic aneurysms
(TAAs)
is medial degeneration with extensive loss of elastic lamellar proteins, including fibrillin-1
(Fbn-1)
. TAA in Marfan Syndrome is associated with
Fbn-1
mutations.
Fbn-1
is degraded by membrane-type 1 matrix metalloproteinase
(MT1-MMP)
in vitro. While elevated levels of
MT1-MMP
have been associated with
TAA
, its effect on
Fbn-1
in
TAA
is unknown.

Objectives

We attempted to characterize tissue
MT1-MMP
and
Fbn-1
in normal and
TAA
patients (non-Marfan) to determine if
Fbn-1
degradation is correlated with elevated
MT1-MMP
levels.

Methods

MT1-MMP
and
Fbn-1
levels were measured in
TAA
tissue from patients undergoing repair and in control aorta
(CON)
from patients undergoing coronary artery bypass grafting. Specimens were homogenized, protein was extracted and quantified, and the extracts were analyzed by Western blotting with antibodies to human
MT1-MMP
and
Fbn-1
.

Results

Elevated levels of
MT1-MMP
were detected in 11/13
TAA
, while 4/6
CON
had undetectable levels (p < 0.02)
(Fig. 1a)
. In preliminary subset analysis,
Fbn-1
degradation products were detected in 2
TAA
samples with high levels of
MT1-MMP
but no detectable
Fbn-1
degradation products were found in 2
CON
samples that showed low levels of
MT1-MMP
(Fig. 1b)
.

Conclusions

Elevated levels of
MT1-MMP
were associated with
Fbn-1
degradation, this may represent a pathologic mechanism of
TAA
formation.
Abstract 515: Anti-Inflammatory Cytokine Interleukin-19 Suppresses the Formation and Progression of Experimental Abdominal Aortic Aneurysms
Authors: Tanaka Hiroki , Xu Baohui , Xuan Haojun , Fujimura Naoki , Wang Wei , Glover Keith J , Iida Yasunori , Michie Sara A , Dalman Ronald L
Published: March 2022

Inflammation plays an important role in the progression of abdominal aortic aneurysms (AAAs). Interleukin (IL)-19 is an anti-inflammatory cytokine that have been show to inhibit inflammatory process. However, its pathogenic role in AAAs is unknown. This study evaluated the effect of IL-19 treatment on the formation and progression of AAAs using transient intra-aortic infusion of porcine pancreatic elastase (PPE) in male C57BL/6 mice. Treatment efficacy was analyzed via serial in vivo transabdominal ultrasound measurements of aortic diameters and histology at sacrifice. PPE infusion significantly increased aortic IL-19 message and protein expression as well as aortic diameter through day 14 in mice. In contrast, exogenous IL-19 therapy initiated one day prior to PPE infusion significantly suppressed subsequent aortic enlargement. Additionally, IL-19 treatment initiated four days following PPE infusion limited further progression of existing aneurysms. Exogenous IL-19 treatment, initiated either pre- or post-PPE infusion, was associated with retained medial smooth muscle cellularity and elastin fibers and attenuated mural inflammation (including reduced T & B cell and macrophage infiltration) in developing aneurysms. In conclusion, IL-19 limits the formation and progression of experimental AAAs.