bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 367: Eph-B4 Mediates Arteriovenous Fistula Maturation via Akt-1
Authors: Foster Trenton , Protack Clinton , Hashimoto Takuya , Bai Hualong , Hanisch Jesse , Hu Haidi , Wang Mo , Santana Jeans , Dardik Alan
Published: June 2021

Objectives:
Although the arteriovenous fistula (AVF) is the gold standard for dialysis access, low rates of fistula maturation prevent optimal fistula use. We have previously shown that stimulation of Eph-B4 in vein grafts prevents wall thickening during venous remodeling. Since Akt-1 is a downstream mediator of Eph-B4 signaling in endothelial cells, we hypothesized that Akt-1 mediates Eph-B4 function during AVF maturation.

Methods:
The infrarenal aorto-caval AVF model was created in wild type C57BL/6 mice and Akt-1 knockout mice as previously described. To stimulate Eph-B4 activity, mice were treated with either EphrinB2/Fc (20 μg IP) or a control vehicle at 48 hr intervals over a 21 day study period. Fistula maturation was monitored with weekly ultrasound measurements; AVF were harvested at day 21 and infrarenal IVC wall thickness was measured using computerized morphometry. Eph-B4, phospho-tyrosine, and Akt-1 immunoreactivity were measured with immunofluorescence.

Results:
In WT mice, Eph-B4 expression increased 4.6-fold after AVF compared to sham operation (p=0.007, t-test). IF revealed unmerged Eph-B4 and p-tyr signal at days 7 and 21. Stimulation of Eph-B4 in WT mice increased Eph-B4 and p-tyr merged signal 6-fold (p=0.005, ANOVA) at 21 days and resulted in a 49% decrease in phospho-Akt-1 signal (p=0.0013, t-test). Eph-B4 stimulation in WT mice was associated with reduced fistula diameter (45% vs. 98.6%; p<0.05, ANOVA) and reduced fistula wall thickness (8.9μm vs. 20.0μm; p=0.0261, t-test). However, in Akt-1 KO mice, Eph-B4 stimulation resulted in no change in fistula diameter (38% vs 22%; p=0.09, ANOVA) and no change in fistula wall thickness (15.3 vs 17μm; p=0.46, t-test).

Conclusion:
There is increased Eph-B4 expression in an AVF, although the Eph-B4 is not phosphorylated. Stimulation of Eph-B4 increases the percentage of phosphorylated Eph-B4 resulting in reduced Akt-1 phosphorylation with consequent reduced fistula diameter and wall thickness. Akt-1 knockout abolishes the effects of Eph-B4 stimulation on wall thickness and diameter. These findings suggest Eph-B4 is an inhibitor of Akt-1 activity and that Akt-1 mediates the effect of Eph-B4 on venous adaptation to the arterial environment.
Abstract 372: The Endothelial Glycocalyx Promotes Homogenous Blood Flow Distribution within the Microvasculature
Authors: McClatchey P Mason
Published: June 2021

Introduction:
Impaired tissue oxygenation is observed in many disease states including congestive heart failure, diabetes, cancer and aging. Decreased tissue perfusion and heterogeneous distribution of blood flow in the microvasculature contributes to this pathology. The physiological mechanisms regulating homogeneity/heterogeneity of microvascular perfusion are presently unknown. We hypothesized that microfluidic properties of the glycocalyx would promote perfusion homogeneity.

Methods:
To test our hypothesis, we used established empirical formulations for modelling blood viscosity in vivo (blood vessels) and in vitro (glass tubes). We first assess distribution of blood flow in idealized arteriolar networks. We next simulated distribution of blood flow at an idealized capillary bifurcation. Finally, we simulated velocity profiles and pressure gradients within the vessel lumen with varying glycocalyx properties using a computational fluid dynamics approach.

Results:
We found that transit time heterogeneity (as assessed by STD to mean ratio) was increased approximately 9x (6.9x-10.6x) using in vitro formulations of blood viscosity relative to in vivo formulations. This effect was mathematically accounted for by increased effective blood viscosity in smaller arterioles. We also found that distribution of blood flow at an idealized microvascular bifurcation was more symmetric using the in vivo formulation than the in vitro formulation (approximately 2x greater disparity between flow in downstream vessels). This effect was mathematically accounted for by an increased hematocrit dependence of blood viscosity. Both the diameter- and hematocrit-based changes in blood viscosity were entirely predictable from fluid dynamics simulations incorporating a space-filling, semi-permeable glycocalyx layer.

Summary:
Our simulations indicate that the mechanical properties of the endothelial glycocalyx promote homogeneous microvascular perfusion.

Conclusions:
The literature provides evidence of both glycocalyx degradation and impaired tissue perfusion in the same disease states. Preservation or restoration of normal glycocalyx properties may be a viable strategy for improving tissue perfusion in a wide variety of diseases.
Abstract 386: Sonic Hedgehog Promotes Stem Cell Differentiation to Vascular Smooth Muscle
in vitro
Authors: Alshamrani Maryam , Fitzpatrick Emma , Redmond Eileen M , Cahill Paul A
Published: June 2021

Background:
The morphogen Sonic Hedgehog (SHh) and its signaling pathway components are significantly up-regulated within adventitial and medial segments from arteriosclerotic vessels in mice concomitant with enhanced accumulation of SMCs. This vessel remodelling is attenuated in vivo following Hh receptor, Patched 1, depletion. There is evidence supporting a role for stem cell-derived vascular smooth muscle (vSMCs) in contributing to arteriosclerotic vascular disease. In this context, SHh signaling may be an important regulator of stem cell self-renewal and differentiation to SMC in vitro.

Aim:
Determine the effects of SHh on bone-marrow derived mesenchymal stem cell (MSC) differentiation to SMC in vitro.

Methods:
Murine CD44+ bone-marrow derived MSCs and Sca1+ rat adventitial progenitor stem cells (APCs) were examined for SHh components and their capacity to differentiate to SMCs before and after treatment with sonic hedgehog (rSHh, 0.5 μg/ml) for 7 d, in the absence or presence of Hh inhibitors cyclopamine (10μM) or HPI-4 (50μM). The transition to SMC was determined be examining intermediate (calponin1, CNN1) and late (myosin heavy chain, Myh11) SMC differentiation marker expression by western blot analysis and immunocytochemistry, respectively.

Results:
Hh signaling components were present on MSCs and APCs. Stem cell growth was unaffected by treatment with Hh inhibitors cyclopamine or HPI-4 at concentrations that inhibited Gli signalling in vitro. Recombinant SHh increased SMC differentiation marker protein protein expression after 7 days, an effect that was inhibited following SHh inhibition with smoothened inhibitors cyclopamine and HPI-4.

Conclusion:
in the absence of any effect on cell growth, Sonic Hedgehog controls mesenchymal stem-like cell differentiation to SMC.
Abstract 387: Regulation of Apolipoprotein B Secretion by Hepatic Sortilin is Dependent on Secretory Stress
Authors: Conlon Donna , Rodrigues Amrith , Strong Alanna , Hand Nicholas , Rader Daniel
Published: June 2021

Sortilin is a multi-ligand sorting receptor involved in trafficking of proteins from the Golgi apparatus to the lysosome and has been widely shown to be associated with plasma lipid traits and coronary artery disease. While over expression of sortilin in the liver reduces VLDL production, the reported effects of the genetic loss of sortilin on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing; loss of sortilin has been shown in different studies to result in both increased and decreased apoB/VLDL secretion. These conflicting studies were carried out in a variety of different models and used different methods of knocking down sortilin expression. To attempt to further clarify the role of sortilin, we explored the role of sortilin deficiency on apoB secretion in the hepatocyte by utilizing 2
in vitro
models of sortilin knockdown; primary hepatocytes from
Sort1-/-
mice and siRNA -treated McA-RH7777 (McA) cells. In both primary hepatocytes and McA cells, loss of sortilin alone was not associated with any change in apoB secretion. The previously reported increases in VLDL secretion occurred on either the background of apoB over expression or in livers of mice on a high fat diet, suggesting the requirement for a metabolic stress. We found that apoB secretion was increased with
Sort1
knockdown as compared to control in isolated primary hepatocytes from Apobec1-/-; hAPOB Tg mice and McA cells stably over expressing apoB. We then sought to increase apoB secretion by lipid loading with oleic acid (OA). While OA increased apoB secretion in all cells, there was no effect of
Sort1
knockdown in this context. However, when the cells were further treated with either palmitic acid, proteasomal inhibitors, or tunicamycin (an ER stress inducer), there was an observed increase in apoB secretion with
Sort1
knockdown, suggesting that sortilin regulates apoB secretion only when both apoB secretion is increased and the cell is stressed. Based on this data, we propose that hepatic sortilin regulates the post-ER fate of apoB for degradation and export and acts to coordinate intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi and the level of post-ER pre-secretory proteolysis activity.
Abstract 388: Impact of Individual Acute Phase Serum Amyloid A Isoforms on HDL Metabolism in Mice
Authors: De Beer Maria C , Kim Myung-Hee , Wroblewski Joanne M , Charnigo Richard C , Ji Ailing , Webb Nancy R , De Beer Frederick C , Van der Westhuyzen Deneys R
Published: June 2021

The acute phase reactant serum amyloid A (SAA) is an HDL apolipoprotein that exhibits biological activities as a pro-inflammatory mediator, but its physiological function(s) are poorly understood. Possible functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are also unclear. Mice deficient in either SAA1.1 or SAA2.1 were used to investigate SAA isoform plasma clearance rates and effects on HDL structure, composition and apolipoprotein catabolism. The absence of either isoform did not affect the size of the normally enlarged HDL found in acute phase wild type mice, and did not result in significant changes in HDL lipid composition. Plasma clearance rates of normal and acute phase HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCR’s) of apoA-I, apoA-II and SAA were distinct, indicating that neither normal nor acute phase particles are cleared as intact particles. No significant difference was found between the FCR’s of SAA1.1 and SAA2.1 in acute phase mice, suggesting that the selective deposition of SAA1.1 observed in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. In the absence of the HDL receptor SR-BI, the clearance rate of SAA was reduced by about 30% and remained significantly greater compared to that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA clearance. These studies contribute to our understanding of the metabolism of SAA and its effects on acute phase HDL composition and catabolism.
Abstract 395: High-performance Gel Permeation Chromatography Analysis Characterizes Lipoprotein Profiles in Patients with Cholesteryl Ester Transfer Protein Deficiency
Authors: Okada Takeshi , Ohama Tohru , Okazaki Mitsuyo , Matsuda Hibiki , Sairyo Masami , Zhu Yinghong , Kawase Ryota , Masuda Daisaku , Koseki Masahiro , Nishida Makoto , Sakata Yasushi , Yamashita Shizuya
Published: June 2021

Introduction:
Recent studies have suggested that HDL efflux capacity could be a potentially beneficial assay to predict the risk of cardiovascular diseases (CVDs), compared to HDL cholesterol (HDL-C) levels. Recently, we developed a novel methodology using high-performance gel permeation chromatography (HPLC) to evaluate lipoprotein particle number of each subclass. In this study, we investigated CETP-deficient (CETP-D) patients, who are elevated in HDL-C levels, but not likely to be protected from atherosclerosis as we reported previously.

Hypothesis:
To evaluate lipoprotein particle number of each subclass may be also useful to predict the risk of CVDs.

Methods:
Nine CETP-D patients, whose serum CETP mass was less than 0.1μg/mL, were compared with 9 normolipidemic controls. Free cholesterol, cholesteryl ester, triglyceride and phospholipid levels in each 20 lipoprotein subclass were determined by computer-assisted HPLC. Furthermore, we calculated the particle number of each subclass by using HPLC data and serum apolipoprotein levels, which is newly-developed LipoSEARCH® system (Skylight Biotech Inc, Akita).

Results:
As we reported previously, serum HDL-C levels were markedly elevated in CETP-D patients compared with controls. The number of very large and large HDL particles in CETP-D patients was markedly higher than that in controls (4237.3±2353.4nM vs 213.4±55.7nM, 7672.2±1368.3nM vs 1720.2±536.6nM, respectively; p<0.001), while the number of small and very small HDL, which have anti-atherogenic function, was significantly lower (4339.1±937.4nM vs 5690.3±467.8nM, 1999.4±514.8nM vs 3256.5±294.0nM, respectively; p<0.001). The number of large and medium LDL was significantly lower in CETP-D patients than that in controls (158.3±36.4nM vs 240.6±51.1nM, 349.1±69.9nM vs 557.3±94.8nM, respectively; p<0.001), whereas the number of very small LDL, which is known to be atherogenic, was significantly higher (233.2±64.8nM vs 171.4±22.1nM, p=0.016).

Conclusions:
We have investigated particle numbers of each lipoprotein subclass in CETP-D patients. Our data might indicate that CETP deficiency leads to pro-atherogenic lipoprotein profile.
Abstract 40: Prothrombotic Role of Platelet Tlr2 in Hyperlipidemia
Authors: Biswas Sudipta , Xin Liang , Panigrahi Soumya , Zimman Alejandro , Yakubenko Valentin , Byzova Tatiana , Salomon Robert , Podrez Eugene
Published: June 2021

A prothrombotic state and increased platelet reactivity are common in hyperlipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products including hydroxy-w-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid can also be modified by hydroxy-w-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PE) accumulate in plasma of hyperlipidemic

ApoE
-/-

mice. CAP-PE directly bind to TLR2 and induce platelet integrin alpha
2b
beta
3
activation and P-selectin expression in TLR2 dependent manner. Platelet activation by CAP-PE includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of TRAF6. This in turn activates the Src family kinases, Syk and PLC gamma 2 and platelet integrins. By intravital thrombosis studies we have demonstrated that CAP-PE accelerate thrombosis in TLR2 dependent manner. Furthermore, we demonstrate that TLR2 deficient mice are protected from accelerated thrombosis induced by hyperlipidemia. Taken together, our studies demonstrate a cross-talk between innate immunity and integrin activation signaling pathways in platelets and reveal that TLR2 plays a key role in platelet hyperreactivity and prothrombotic state in hyperlipidemia.
Abstract 401: Functional Analysis of Coronary Artery Disease-associated PLPP3 Gene Variants
Authors: Morris Andrew
Published: June 2021

Heritable variants of the PLPP3 gene are associated with human coronary artery disease risk. Expression of the PLPP3 gene product, lipid phosphate phosphatase 3 (LPP3) is strongly increased in blood and vascular cells by pro-atherogenic, pro-inflammatory stimuli including oxidized LDL. PLPP3 is essential for vascular development in mice but animals with inducible post-natal or tissue specific inactivation of the gene exhibit accelerated development of aortic atherosclerosis. These observations suggest the hypothesis that PLPP3 normally functions to suppress the development and progression of atherosclerosis. The coronary artery disease-associated PPAP2B haplotype block is in the final intron of the gene. Recently published analysis of chromatin remodeling in oxidized LDL stimulated human monocytes identifies dynamic changes in these coronary artery disease associated loci. We show that several of these loci correspond to potential or identified regulatory genomic elements. Functional analysis of these elements identifies multiple cFos/AP1 and cEBP regulated transcriptional enhancer sequences that exhibit allelic variation that could impact on their function as regulators of PLPP3 gene expression. Some of these enhancer sequences bind their transcription factor targets in an allele specific manner and are functional activators of transcription from the PLPP3 promoter. Because haplotypes containing the coronary artery disease associated enchancer sequence variants are also associated with attenuated increases in PPAP2B expression in oxidized LDL treated human monocytes our observations support the hypothesis that heritable cardiovascular disease risk is associated with attenuation of cis acting enhancer sequences that normally promote LPP3 expression. These observations also raise the possibility that these intronic enhancers have longer distance or trans-acting effects on other genes that also contribute to coronary artery disease risk
Abstract 416: Evaluation of Antibody Responses Toward Post-Translationally Modified and Unmodified Peptide Epitopes of Apolipoprotein A-I
Authors: Henson David , Venditto Vincent J
Published: June 2021

Antibody responses targeting ApoA-I have been documented in patients suffering from chronic inflammation associated with obesity, autoimmunity and cardiovascular disease (CVD) and correlate with a poor prognosis. Patients presenting with an acute myocardial infarction (AMI) have measurable IgG targeting the lecithin:cholesterol acyltransferase (LCAT) domain of ApoA-I and are shown to bind an epitope containing an oxidized methionine (Met-148(O)). Antibodies targeting ApoA-I are thought to be serum markers for CVD and not responsible for disease progression, but their exact role is poorly understood. We hypothesize that the B cell response to ApoA-I is nuanced with different isotypes of both protective and pathologic antibodies induced during the course of disease. To investigate this hypothesis, we synthesized a series of unmodified and modified peptides corresponding to the LCAT domain of mouse, monkey and human ApoA-I for ELISA and immunization studies. ELISA with commercially available goat polyclonal antibodies induced by immunization with human ApoA-I suggest that the LCAT domain possesses immunogenic properties and the antibodies are capable of binding to the unmodified and Met-148(O) peptides. Although affinity to the unmodified peptide appears to be higher by ELISA, pre-incubating the polyclonal antibodies with a solution of the Met-148(O) peptide significantly inhibits binding to the unmodified peptide. These data suggest that the oxidized peptide in solution may undergo a structural change that increases antibody affinity. Structural analysis of the peptides are ongoing. Serum analysis from patients presenting with an AMI and 30 days after cardiac event show a decline in IgG responses toward the Met-148(O)-modified peptide during that period. Analysis of monkey serum provides a unique comparison. Monkeys have a methionine to valine mutation at position 148, which results in a non-oxidizable residue, and instead show a decrease in IgG toward the unmodified sequence when on an atherogenic diet for 12 weeks. Correlating these ELISA assays with atherosclerosis observed in the patients and macaques along with ongoing
in vivo
immunization studies in mice will generate insight into the nuanced B cell response toward ApoA-I during CVD.
Abstract 442: Generation of an Abcg1 Knock Out Mouse on the Reversa Background
Authors: Douglas Gillian , Trelfa Lucy , Channon Keith , Davies Ben , Bhattacharya Shoumo
Published: June 2021

Background:
Clinically HDL mediated reverse cholesterol transport (RCT) from macrophages has been shown to be inversely associated with carotid intima media thickness. Cholesterol efflux to mature HDL is mediated by ATP binding cassette transporter G1 (Abcg1). Abcg1 pays a key role in cholesterol transport with loss of function in macrophages and endothelial cells associated with significant cholesterol accumulation. However, mechanistic studies into the role of Abgc1 in plaque regression have been restricted due to the limitations of current regression models.

Aims:
To use TALENS mediated genomic editing to generate an Abcg1 knockout mouse on the REVERSA background to enable the investigation of its role in plaque regression.

Methods and results:
TALENs constructs were targeted to exon 3 upstream of the phosphate binding Walker A domain. TALEN mRNA was injected into REVERSA oocytes which were then implanted into foster mice. Founders were screened by Cel1 nuclease assay and sequencing. Three independent alleles were identified two of which create frameshift mutations (predicted to be null alleles) and one which resulted in a 3 amino acid deletion and a one amino acid substitution near the Walker A domain (potential hypomorphic allele). The two founder lines with frame shift mutations (KO 145 and 171) were taken forward for additional analysis. RNA extracted from primary macrophages from WT (REVERSA) and homozygous Abcg1 knockout mice was used to confirm the mutation was transcribed to RNA. Intron-spanning primers were designed and a product of the expected size was obtained and sequence analysis confirmed the insertion (KO-145) and deletion (KO-171) within the WALKER A domain of Abcg1. To ensure the mutations resulted in loss of function, a radioactive RCT assay was carried out in bone marrow derived primary macrophages. A significant decrease in RCT to HDL was observed in macrophages from both the KO-145 and KO-171 lines as expected there was no difference in RCT to ApoA1-I.

Conclusions:
Using genomic editing we have generated a gene specific knockout on the REVERSA background which will enable, for the first time, the investigation of the role of Abcg1 in plaque regression.
Abstract 456: Is There a Relationship Between an Elevated Coronary Calcium Score and the Incidence of Kidney or Gallbladder Calculi
Authors: Kumar Vishesh , Kelly Shawn , Raizada Amol , Anuwatworn Amornpol , Yee Jimmy , Thompson Paul , Stys Adam
Published: June 2021

Background:
Elevated calcium score is a marker of increased risk in the development of cardiovascular and cerebrovascular disease. It is unknown whether an elevated coronary artery calcium score is associated with extravascular calcium deposition in disease states such as nephrolithiasis and cholelithiasis. The aim of this study was to explore this relationship in asymptomatic patients who had elective coronary calcium scoring performed at the University of South Dakota Sanford Medical Center.

Methods:
We performed a retrospective review of data from 38,546 consecutive patients from the coronary calcium screening database. Patients with a history of calculi were identified using ICD-9 codes for calculus of kidney and ureter (592) and cholelithiasis (574). Cases were analyzed according to gender and age groups, and proportions calculated according to calcium score tertile.

Results:
The results are summarized in Table 1. Overall, the proportion of patients with calculi increases with each rise in the coronary artery calcium score. However, this relationship is best demonstrated in the elderly male and younger female populations. This relationship was further analyzed using a chi-square contrast test. The results, as described in Table 2, indicate that there is a significant relationship between elevated coronary calcium and likelihood of calculi conditions in young females (p=0.0040), however, the proportion in elderly males does not exhibit a statistical difference in proportion (p=0.1143).

Conclusion:
Elevated coronary artery calcium is associated with an increased likelihood of having a diagnosis of extravascular calculi development such as nephrolithiasis or cholelithiasis. Younger females appear to best demonstrate this relationship. Although the proportions are small, further studies are needed to substantiate a biochemical basis for this relationship.
Abstract 457: Transcription Coactivator MED1 Protects Against Atherosclerosis by Modulation of Macrophage Polarization
Authors: Bai Liang , Liu Enqi
Published: June 2021

Mediator 1 (MED1), a key subunit of the Mediator complex, interacts with several nuclear receptors and transcription factors to direct gene-specific transcription. It is well-known that MED1 play important roles in lipid metabolism. However, the role and underlying mechanisms of MED1 in atherosclerosis remains unclear. Here, macrophage-specific MED1 knockout (MED1
ΔMac
) mice were generated to investigate the contribution of MED1 on atherogenesis in vivo. We demonstrate that among mice deficient in apolipoprotein E (Apoe), the additional loss of macrophage MED1 (MED1
ΔMac
/ApoE
-/-
) exhibited significantly larger atherosclerotic lesions in the whole aortic tree and aortic root compared with MED1
fl/fl
/ApoE
-/-
littermates on either the chow or the western diet, and these effects were also found in low-density lipoprotein (LDL) receptor-deficient (LDLR
-/-
) mice reconstituted with bone marrow from MED1
ΔMac
mice. Peritoneal macrophages from MED1
ΔMac
/ApoE
-/-
mice had significantly increased expression of gene markers for M1-like macrophages, including IL-1β, IL6,COX2, iNOS, Gro1, MCP1 and TNFα, etc, whereas decreased levels of anti-inflammatory genes for M2-like macrophages, such as Arg1, Mrc1, Retnla, Chi3l3 and PPARγ. Over-expression of MED1 using adenovirally-driven MED1 (Ad/MED1) in MED1
fl/fl
/ApoE
-/-
macophages repressed the proinflammatory gene expression. Re-expression of MED1 using Ad/MED1 counteracted the high level of inflammatory gene in MED1
ΔMac
/ApoE
-/-
macophages. Furthermore, gene expression profiling and PCR array showed that MED1-deficient macrophages exhibited the increased M1 targets and decreased M2 targets. These data demonstrate that MED1 expression by macrophages has anti-atherogenic effects via regulation of macrophage polarization. MED1 may be considered as a potential therapeutic target to treat atherosclerosis.
Abstract 458: Fcgamma Receptor IV Expressed on Inflammatory Cells Drive Diet-induced Atherosclerosis in Hyperlipidemic Mouse Model
Authors: Popovic Branimir , Feck Doug , Nagarajan Shanmugam
Published: June 2021

Objective:
Functionally, Fcgamma receptors (FcgRs) can be classified as activating (FcgRI, III, and IV) and inhibitory (FcgRII) receptors. We have reported that combined deficiency of all three activating FcgRs in apoE knockout mice decreased atherosclerosis. In this report, we investigated the independent role of FcgRI and FcgRIV in the progression of atherosclerosis. We investigated the hypothesis that the deficiency of FcgRIV, one of the activating FcgRs, inhibits atherosclerosis in a hypercholesterolemic mouse model. We tested the hypothesis that FcgRI and FcgRIV exacerbate atherosclerosis using apoE-FcgRI dKO and apoE-FcgRIV deficient mice.

Approach and Results:
ApoE-FcgRI and apoE-FcgRIV double knockout mice (dKO) congenic to the C57BL/6 were generated and atherosclerotic lesions were assessed. Our results show that arterial lesions were not different between apoE-FcgRI dKO and apoE knockout (apoE KO) mice. Interestingly, arterial lesions were significantly decreased in a regular chow or a high-fat diet fed apoE-FcgRIV dKO male and female mice, relative to apoE KO mice. Bone marrow chimeras were used to address the relative contribution of FcgRIV expressed on hematopoietic cells including macrophages and dendritic cell. ApoE KO mice transplanted with apoE-FcgRIV dKO marrow showed significantly reduced arterial lesions relative to recipient mice transplanted with apoE KO marrow. Next, we investigated whether pro-inflammatory response contributed to the pro-atherogenic effect of FcgRIV. Activated CD4+ T cells of apoE-FcgRIV dKO mice showed increased secretion of IL-10, whereas IFN-gamma and IL-17 by T cells were decreased. Interestingly, dendritic cells at the lesion-prone vascular site from apoE-FcgRIV dKO mice induced increased IL-10 secretion by LDL-specific T cells. Moreover, FcgRIV KO and apoE-FcgRIV dKO macrophages showed decreased inflammasome activation as evidenced by decreased IL-1 beta response.

Conclusions:
Our findings demonstrate that the pro-inflammatory responses initiated by FcgRIV, one of the activating FcgRs, contribute to the progression of atherosclerosis.
Abstract 466: Therapeutic Immunization with an Antiglycosaminoglycan Antibody Reduces Atherosclerotic Lesion Progression in Apolipoprotein E-Deficient Mice
Authors: Sarduy Roger , Brito Victor , Soto Yosdel , Delgado-Roche Livan , Griñán Tania , Viera Justo , González Jordan , Marleau Sylvie , Vázquez Ana M
Published: June 2021

Subendothelial retention of apoB-containing lipoproteins by interaction with glycosaminoglycan-side chains of proteoglycans is considered the key initiating step of atherogenesis. Previously, we characterized the antiatherogenic properties of the chimeric mAb chP3R99, which binds sulfated GAG, inhibits LDL-chondroitin sulfate (CS) association, and abrogates LDL oxidation in vitro and in vivo. In preventive settings, rabbits and mice immunized with this mAb showed reduced atherosclerotic lesions, related with the induction of anti-chondroitin sulfate (CS) antibodies. Now we focus in define the immunization schedule which can induce in apolipoprotein E-deficient (apoE-/-) the highest anti-CS antibody response and the greater reduction of atherosclerotic lesion progression. ApoE
-
/
-
mice (6-8 wk. old) fed a chow diet received four s.c. injections of 50 or 200 μg of chP3R99 mAb. Autologous antichondroitin sulfate (CS) antibody response was evaluated in sera by ELISA. Mice who were immunized with 200 μg generated a significant higher response against CS than the ones that received 50 μg. Then, to evaluate the association of the induction of a higher anti-CS response in the atherosclerotic lesion progression, apoE-/- mice fed with a high-fat high-cholesterol diet from 4 to 18 wk. of age, received 6 doses of 50, 100 or 200 μg of the mAb starting when 5% of the aortic area was covered by lesions. Animals were sacrificed and aortas isolated to determine the presence of lesions by histologic studies. Mean aortic lesion areas of 50 and 100 μg chP3R99-treated mice were significant reduced by ~40% in comparison with mice treated with an isotype-matched control mAb. In contrast, 62% reduction in total lesion area was observed in mice treated with 200 μg of chP3R99. Again, there was an association between the level of anti-CS antibody response and the reduction of atherosclerotic lesion progression. In conclusion, this study demonstrated the dose dependence of the anti-CS antibody response and its relationship with the arresting of the atherosclerotic progression induced by chP3R99 mAb immunization. Our results also supports the potential use of this antiglycosaminoglycan antibody-based immunotherapy as a novel approach to target advanced atherosclerosis
Abstract 501: A Pivotal Role of Smooth Muscle Bmal1 in Mineralocorticoid Receptor Agonist plus Salt Induced Mouse Aortic Aneurysm
Authors: Lutshumba Jenny , Liu Shu , Guo Zhenheng , Gong Ming C
Published: June 2021

Rationale:
Bmal1 is a basic helix loop helix (bHLH) transcription factor and is an indispensable core clock gene. Bmal1 is expressed in vascular smooth muscle and has been implicated in atherosclerosis and vascular remodeling. However, it is unknown whether vascular smooth muscle Bmal1 is involved in aortic aneurysm. We use a smooth muscle selective Bmal1 knockout (SM-Bmal1
-/-
) mouse and mineralocorticoid receptor agonists plus salt induced aortic aneurysm mouse model to address this specific question.

Approach and Results:
Aortic Aneurysm was induced in SM-Bmal1
-/-
and littermate controls by subcutaneously implanting a 50mg DOCA (deoxycorticosterone acetate) pellet for 21 days or mini-pump with 200μg/Kg/day aldosterone for 28 days and drinking 0.9% saline solution. The abdominal aortic aneurysm incidence was dramatically decreased from control 30.7% (4-month-old) and 68.0% (8-month-old) to 4.0% (p<0.05) and 0.0% (p<0.001) in SM-Bmal1
-/-
mice respectively. This was associated with a significant decrease in the aortic inner and external diameters. While SM-Bmal1 deletion did not alter the plasma sodium concentration, it significantly decreased the elastin breakage in aorta. In situ zymography assay demonstrated a significantly suppressed gelatinase activity in SM-Bmal1
-/-
mouse aorta when the metalloproteinases (MMPs) were complexed with their tissue inhibitors (TIMPs). Gel zymography showed no decrease in MMP2/9 activities in the SM-Bmal1-KO mouse aorta, real-time PCR analysis showed TIMP4 mRNA and immunohistochemistry analysis showed TIMP4 protein were selectively up-regulated among the four TIMP isoforms. Mouse TIMP4 promoter contains 7 E-boxes, ChIP assay of the mouse aorta tissues showed 4 of them bound to Bmal1. Further TIMP4 promoter luciferase activity analysis demonstrated TIMP4 promoter activity was dramatically activated by the deletion of Bmal1.

Conclusions:
Deletion of Bmal1 from smooth muscle protects mice from mineralocorticoid receptor agonist plus salt induced aortic aneurysm at least in part via up-regulating TIMP4.
Abstract 503: Chronic Kidney Disease is a Risk for Abdominal Aortic Aneurysm but Diabetes Mellitus is a Protective Factor in Japanese
Authors: Takeuchi Hidemi , Uchida Haruhito , Okuyama Michihiro , Umebayashi Ryoko , Kakio Yuki , Okuyama Yuka , Wada Jun
Published: June 2021

Objective:
Abdominal aortic aneurysm (AAA) is the most common aortic aneurysm. Chronic kidney disease (CKD) and diabetes mellitus (DM) are considered as risk factors for cardiovascular diseases. However, the association between CKD and AAA remains unknown. Although DM has been reported to exert protective effect on the incidence and development of AAA in western population, such protective role of DM was not explored in the Asian population. The purpose of this study was to determine the relationship of CKD and DM and the presence of AAA. We performed a cross-sectional retrospective case-control study in Asian population.

Methods and Results:
We enrolled 261 patients with AAA (AAA+) and age-and-sex matched 261 patients without AAA (AAA-) at two hospitals between 2008 and 2014, and examined the association between the risk factors and the presence of AAA. Furthermore, to investigate AAA prevalencein each group, we enrolled 1126 patients with CKD and 400 patients with DM. The presence of CKD in patients with AAA+ was significantly higher than that in patients with AAA- (AAA+; 65 %, AAA-; 52 %,
P
= 0.004). The presence of DM in patients with AAA+ was significantly lower than that in patients with AAA- (AAA+; 17 %, AAA-; 35 %,
P
< 0.001). A multivariate logistic regression analysis demonstrated that hypertension, ischemic heart disease and CKD were independent determinants, whereas, DM was the only independent protective factor, for the presence of AAA. The prevalence of AAA in patients with CKD 65 years old and above was 5.1 %, whereas, that in patients with DM 65 years old and above was only 0.6 %.

Conclusion:
CKD is a positively associated with the presence of AAA. In contrast, DM is a negatively associated with the presence of AAA.
Abstract 511: Adverse Outcomes in Patients with Diabetes Mellitus Following Stenting for Lower Extremity Peripheral Arterial Disease
Authors: Kiani Sarah , Salahuddin Usman , Slaughter Haekyung Jeon , Mohammad Atif , Brilakis Emmanouil S , Banerjee Subhash
Published: June 2021

Introduction:
There is limited data on outcomes of percutaneous endovascular intervention for lower extremity peripheral artery disease (PAD) in patients with diabetes mellitus (DM). We assessed the hypothesis that patients with DM, in comparison to patients without DM, have higher rates of major adverse cardiovascular and limb events after lower extremity PAD stenting.

Methods:
1,006 patients with primary stent implant procedures between January 2005 and October 2015 enrolled in the observational XLPAD registry (NCT01904851) were analyzed for 12 month major adverse cardiovascular events (MACE; all-cause death, myocardial infarction, and stroke) and major adverse limb events (MALE; target limb repeated endovascular intervention, surgical revascularization, and major amputation). Cochran-Mantel-Haenszel statistics was used for overall association of categorical baseline characteristics; Cox proportional regressions and Kaplan-Meier curves were used for median time to event analysis.

Results:
At baseline, patients with DM had higher prevalence of coronary artery disease (74.9% vs. 56.2%; p<0.0001), heart failure (22.9% vs. 10.9%; p<0.001), prior myocardial infarction (27.3% vs. 22.5%; p=0.0076) and prior stroke (10.3% vs. 5.8%; p<0.0001) in comparison to patients without DM. Cox proportional regressions after adjusting for baseline characteristics showed significantly higher MACE (8.5% vs. 4.0%; Hazard ratio (HR) 1.99; 95% CI 1.26-3.50; p=0.003,
Figure 1A
) as well as MALE (49.0% vs. 40.9%; HR 1.22; 95% CI 1.01-1.48; p=0.043,
Figure 1B
) in patients with DM at 12-months.

Conclusion:
PAD in patients with DM is associated with significantly higher rates of major adverse cardiovascular and limb events.
Abstract 522: Cd163 Macrophage Content and Glucose Transporter-1 Expression in Aspirated Deep Vein Thrombus Are Associated With the Time After Onset
Authors: Yamashita Atsushi , Furukoji Eiji , Moriguchi-Goto Sayaka , Kojima Mio , Sugita Chihiro , Sato Yuichiro , Asada Yujiro
Published: June 2021

Background:
Leukocytes involve resolution of deep vein thrombosis (DVT) and glucose uptake may reflect acute phase of DVT. Macrophages in M2-like phenotype are related to the tissue repair. However, markers reflecting thrombus age have not been established. The aims of this study were to identify a cellular or metabolic marker that reflects the time after onset in human aspirated DVT.

Methods:
We histologically and immunohistochemically analyzed 17 aspirated thrombi. The times from onset to aspiration ranged from 5 to 60 days. Paraffin sections were stained with hematoxylin and eosin and antibodies for fibrin, glycophorin A, integrin α2bβ3, CD68, M2-markers (CD163, CD206), CD34, smooth muscle actin (SMA), glucose transporter (Glut)-1, and hypoxia inducible factor (HIF)-1α (a metabolic regulator).

Results:
All thrombi were immunopositive for glycophorin A, fibrin, and integrin α2bβ3, CD68, CD163, CD206 and Glut-1, and contained granulocytes without lytic change. Almost all of the thrombi had small foci of CD34 or SMA immunopositive areas. The CD163- but not CD206-positive cells existed predominantly among the macrophage population. Glut-1 was exclusively expressed on the erythrocytes. There were a few HIF-1α immunopositive nuclei. The values of the immunopositive areas for glycophorin A and Glut-1 were negatively correlated and those of CD68 and CD163 were positively correlated with the time after the onset. The CD163-positive macrophages were associated with glycophorin A, CD34, or SMA positive cell-rich areas.

Conclusions:
These findings suggest that CD163 and Glut-1 expression could be markers for the evaluations of thrombus age in DVT and CD163 macrophages might play a role in the organization of DVT.
Abstract 529: Assessing Hemostasis and Platelet Function Using a Microfluidic Device Lined with Fixed Human Endothelium
Authors: Jain Abhishek , van der Meer Andries D , Papa Anne-Laure , Barrile Riccardo , Lai Angela , Schlechter Benjamin L , Otieno Monicah A , Louden Calvert S , Hamilton Geraldine A , Michelson Alan D , Frelinger Andrew L , Ingber Donald E
Published: June 2021

Mutual signaling between endothelium and platelets is widely recognized as critical for regulation of hemostasis and thrombotic disorders associated with various diseases, such as, arteriosclerosis, sepsis, and diabetes. Yet no reliable and practical assays exist that can measure the effects of cross-talk between platelets and inflamed vessel walls in the presence of physiological shear. Flow chambers have been applied previously to study basic science, but they have not been used in practical settings due to the difficulty in maintaining living cells in devices. Specifically, because it is extremely difficult to maintain living cell cultures for extended times, it would be impossible to maintain the robustness of these assays. Here, we describe a microfluidic device lined by a chemically fixed human endothelium that retains its ability to support thrombus formation in the presence of blood flow for multiple days under standard storage conditions. We coat the inner surface of a rectangular device with collagen and then culture with endothelial cells on all four walls in order to create a vascular tube. Under activation by increasing doses of TNF-α followed by fixation, the device continues to exhibit multiple proinflammatory and adhesion molecules. Correspondingly, a dose-dependent increase in platelet adhesion to the endothelial layer is also observed. Importantly, we do not observe any significant difference in platelet adhesion between living and fixed endothelium. Furthermore, the morphology of thrombi appears similar to that of thrombi that form on living endothelium of a laser-injured mouse
in vivo
. To establish clinical utility, we also perfused whole blood of patients taking antiplatelet drugs, and found that these subjects showed a significant reduction in platelet adhesion in our devices. In contrast, conventional aggregometry analysis was very time consuming and tedious. Also, on a collagen-coated flow chamber we found no significant difference in platelet coverage between normal controls and patients. Taken together, this study confirms that this microdevice-incorporating fixed endothelium, whole blood and shear stress-can be used as a global hemostasis assay in research as well as many practical and clinical settings.
Abstract 534: Deficiency of Protease-activated Receptor 2 Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm Independent of Vascular Smooth Muscle Cell Tissue Factor
Authors: Hall David E , Mann Adrien , Jones Shannon M , Mackman Nigel , Owens A. Phillip
Published: June 2021

Objective:
Tissue factor (TF) is constitutively expressed in subendothelial cells, including vascular smooth muscle cells (VSMCs), and maintains hemostasis. The TF/factor VIIa complex as well as factor Xa have been shown to activate protease-activated receptor 2 (PAR-2). We and others have shown that TF/FVIIa-PAR-2 signaling induces migration of VSMCs in vitro and this is reduced in TF and PAR-2 deficient cells. We previously demonstrated C57BL/6 mice with 1% of normal TF levels (Low TF mice) had increased suprarenal abdominal aortic diameter and incidence of abdominal aortic aneurysm (AAA) compared to littermate controls. However, the source of cellular TF and whether signaling occurred through PAR-2 has not been examined. Our objective was to determine the importance of VSMC-specific deletion of TF and PAR-2 deficiency on the etiology of AAA.

Methods and Results:
VSMC-specific TF deficiency was evaluated on a low-density lipoprotein receptor deficient (Ldlr
-/-
) background. Male mice were fed a fat-enriched diet (21% milk fat) and infused with angiotensin II (AngII; 1,000 ng/kg/min) for 28 days. Ldlr
-/-
/TF
flox/flox
SM22αCre
+
mice had increased abdominal aortic luminal diameters (Cre
+
: 1.92 ± 0.16 (n = 12); Cre
-
: 1.22 ± 0.03 mm (n = 10); P = 0.001) and incidence of AAA (100% vs. 50%; P = 0.01) compared to Cre
-
littermates. Next, we determined the effects of PAR-2 deficiency in AAA. Ldlr
-/-
/PAR-2
+/+
(n = 12) and Ldlr
-/-
/PAR-2
-/-
(n = 15) male mice were fed a fat-enriched diet and infused with AngII for 28 days. PAR-2 deficiency attenuated abdominal aortic luminal diameters (PAR-2
+/+
: 1.78 ± 0.06 mm; PAR2
-/-
: 1.17 ± 0.03 mm; P = 0.001) and incidence of AAA (84% versus 40%; P = 0.048) when compared to littermate controls. Total plasma cholesterol concentrations, lipoprotein cholesterol distributions, or increased systolic blood pressure was not different between groups.

Conclusion:
These results suggest that TF limits AAA growth and incidence via VSMCs in a PAR-2-independent manner. To verify this hypothesis, studies are currently underway to determine the effect of Factor Xa (FXa) inhibitors in PAR-2 deficient mice.
Abstract 537: Lipoproteins Transport Functional Non-human Small RNAs that Regulate Gene Networks Spanning Inflammation and Lipid Metabolism
Authors: Allen Ryan M , Zhao Shilin , Sheng Quanhu , Linton MacRae F , Vickers Kasey C
Published: June 2021

Cardiovascular disease (CVD) is a significant health and financial burden to our society that demands new and more effective therapies. Although dyslipidemias are primary risk factors for CVD, alternative lipoprotein functions also contribute to CVD and warrant greater understanding. We have found that high-density lipoproteins (HDL) transport microRNAs (miRNA) in circulation and HDL-miRNA signatures are significantly altered in hypercholesterolemia and atherosclerosis. Moreover, we found that HDL transfers extracellular miRNAs to recipient cells where they regulate gene expression through post-transcriptional repression of mRNA targets. We used high-throughput small RNA (sRNA) sequencing to identify and quantify miRNAs and novel sRNAs on HDL and other lipoproteins. Most interestingly, we found that the majority of sRNAs on lipoproteins are likely derived from non-human organisms of multiple kingdoms. Based on these observations, we hypothesized that human and non-human sRNAs on lipoproteins are unique regulators of gene networks that contribute to the complex pathophysiology of CVD. To assess this hypothesis, highly pure lipoproteins (HDL, low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL)) were isolated from plasma of hypercholesterolemic (heterozygous familial hypercholesterolemia, n=9) and healthy (n=7) subjects. We found that HDL, LDL, and VLDL each transport unique sRNA signatures, which are differentially altered in hypercholesterolemic subjects. Using a human tissue library, we identified tissues that likely take up non-human sRNAs. We also found that each class of lipoprotein is capable of transferring non-human sRNAs to multiple cell types, and that transfer efficiency is altered in hypercholesterolemia. Lastly, using a combination of in vitro over-expression and locked-nucleic-acid inhibition for candidate, lipoprotein-enriched, non-human sRNAs, we have discovered novel regulatory networks for critical genes in inflammation and lipid metabolism. This work demonstrates that lipoprotein transport of endogenous and exogenous sRNAs likely have complex roles in the progression and resolution of CVD and are a source of untapped potential for nucleic-acid based therapeutics.