bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 80: Interleukin 15 Plays an Important Role in the Development of Atherosclerosis in Apolipoprotein E Knockout Mice
Authors: Dadoo Omid , Ashkar Ali , Richards Carl , Trigatti Bernardo
Published: July 2021

IL-15 is an essential cytokine for the development of NK and CD8+T cells and also involved in activation of inflammation in macrophages. Therefore, we hypothesized that IL-15 may affect the development of atherosclerosis via regulating immune/inflammatory cells. To test this we generated apoE KO mice that either overexpressed (il-15TgapoE-/-) or lacked either one (il-15+/-apoE-/-) or both copies (il-15-/-apoE-/-) of the IL-15 gene. Deficiency of IL-15 substantially reduced atherosclerosis in spite of increased plasma lipoprotein cholesterol levels. Interestingly, il-15+/-apoE
-/-
mice exhibited intermediate levels of atherosclerosis. Conversely, overexpression of IL-15 in il-15tg apoE
-/-
mice resulted in substantially increased atherosclerosis compared to apoE
-/-
littermates. Deficiency of IL-15 resulted in a lack of NK cells and reduced populations of monocytes and CD8+ T cells. Correspondingly, reduced levels of CD11b+ and CD8a+ cells were detected in atherosclerotic lesions from il-15-/-apoE
-/-
mice compared to those from apoE
-/-
mice. To test the role of NK cells in atherosclerosis, NK1.1+ cells were immune-depleted in apoE
-/-
mice and lesions size was analyzed. Atherosclerosis was reduced but to a lesser extent than in age matched il-15-/-apoE
-/-
mice. Plaques from these mice were almost devoid of CD8+T cells. Deletion of a single copy of the il-15 gene (il-15+/-) in NK/NKT cell immunodepleted apoE
-/-
mice resulted in a further reduction in atherosclerosis, suggesting the involvement NK/NKT and CD8-T cell independent pathways participate in IL-15’s proatherogenic effects. Treatment of macrophages with recombinant IL-15 in vitro induced expression/activity of a variety of pro-inflammatory targets. Furthermore, il-15-/-apoE
-/-
mice exhibited reduced levels of MCP-1 in plaques and reduced circulating levels of SAA and IL-6, whereas circulating levels of SAA and IL-6 were greatly increased in il-15tg apoE
-/-
mice compared to apoE
-/-
littermates. In conclusion IL-15 plays a significant role in promoting atherosclerosis through multiple pathways including promotion of the survival/recruitment of NK/CD8+T cells, as well as the modulation of monocyte levels, and/or direct activation of macrophages.
Abstract 90: The Atheroprotective Effects of Id3 Are Linked to B-1a Cell Proliferation and Plasma Levels of E06
Authors: Perry Heather M , Doran Amanda , Oldham Stephanie N , Gonen Ayelet , Que Xuchu , Tsimikas Sotirios , Witztum Joseph L , Bender Tim , Garmey James , Taylor Angela , McNamara Coleen A
Published: July 2021

Background:
The HLH transcription factor, Id3, is essential for B cell-mediated atheroprotection in mice and a functionally significant single nucleotide polymorphism in the human
ID3
gene at rs11574 is associated with carotid intimal medial thickness in humans. Yet, the mechanisms mediating Id3 atheroprotection are poorly understood. Recent studies provide clear evidence that B cell effects on atherosclerosis are subset dependent; with B2 cells promoting and B-1a cells attenuating atherosclerosis in a sIgM-dependent manner. Innate, natural IgM antibodies, such as E06, recognize oxidized phospholipids, are produced by B-1a cells, and inhibit atherogenesis. Therefore, we hypothesized that Id3 would regulate plasma levels of E06 IgM and atheroprotective B-1a cells.

Methods & Results:

Id3
-/-
ApoE
-/-

mice (n = 9) at 8 weeks old had lower plasma levels of E06 compared to control

Id3
+/+
ApoE
-/-

mice (n = 9) (1500 vs. 2750 RLU, p < 0.05) as measured by ELISA. This was not due to lower total IgM (270 vs. 100 ug/mL respectively, p < 0.05). ApoB-100 levels were not different (1500 vs 1600 RLU, n.s.). Consistent with plasma data, the number of B-1a cells, assessed by flow cytometry, was lower in

Id3
-/-
ApoE
-/-

(n = 5) compared to

Id3
+/+
ApoE
-/-

mice (n = 4) (0.8 x10
5
vs 3.0 x10
5
, p < 0.05). There was no difference in transcript levels, as measured by real-time PCR, of E06 or sIgM in fluorescence-activated cell sorted B-1a cells. Furthermore, we found decreased homeostatic proliferation, measured by
in situ
CFSE dilution, of B-1a cells in

Id3
-/-
ApoE
-/-

mice (n = 5) compared to control mice (n = 5) (30.1% vs. 50.0%, p = 0.001). Additionally, humans with the functionally significant polymorphism in
ID3
had lower plasma levels of IgM to MDA-LDL (n = 97, trend with p = 0.08) and no difference in IgG as measured by ELISA.

Conclusion:
Taken together, these data suggest a novel role for Id3 in regulating B-1a cell proliferation, resulting in reduced plasma levels of the atheroprotective natural antibody, E06. Moreover, the data suggest that the
ID3
polymorphism in humans at rs11574 may link protective IgM to modified lipids with vascular disease.
Abstract 98: Interleukin-1 Beta Deletion Protects Against Abdominal Aortic Aneurysm Development
Authors: Johnston William F , Angulo Morgan S , Su Gang , Zhao Yunge , Bhamidipati Castigliano M , Alexander Matthew R , Owens Gary K , Upchurch Gilbert R , Ailawadi Gorav
Published: July 2021

Objectives:
Inflammation is known to be critical in abdominal aortic aneurysm (AAA) formation. While interleukin-1 beta (IL-1B), a proinflammatory cytokine, is elevated in AAA samples from human and murine samples, the role of IL-1B during AAA formation remains largely unknown. We hypothesized that IL-1B is a significant positive contributor to the AAA inflammatory pathway.

Methods:
Eight to 12 week old male mice deficient in the expression of IL-1B (IL-1B KO; n=9) and their corresponding wild type background mice (WT, n=5) underwent AAA induction by purified porcine elastase (0.4 units) infusion of the infrarenal aorta. After 14 days, the maximum diameter of the infrarenal abdominal aorta was measured, and the aortas were harvested for molecular and histologic analyses, including immunostaining for macrophages, neutrophils, and elastin. Inflammatory and smooth muscle genetic markers were compared using real-time PCR.

Results:
IL-1B KO mice had significantly decreased aneurysm formation when compared to WT controls [34.2% aortic dilation versus 87.2% aortic dilation (p=0.002), respectively]. Immunostaining aortic cross-sections demonstrated markedly less macrophage and neutrophil infiltration of the aortic wall with preservation of elastic lamina in the aortas of IL-1B deficient mice. Genetic expression of inflammatory markers [matrix metalloproteinases (MMP) 2, 3, and 9] were decreased in the aortas from IL-1B KO mice compared to WT mice, while smooth muscle markers (i.e. smooth muscle actin, myosin heavy chain, and smooth muscle 22α) were all significantly greater in the aortas from IL-1B KO group.

Conclusions:
Deletion of IL-1B is protective for AAA development in a murine elastase model and appears to be a critical step in the pathway for AAA formation. IL-1B deletion appears to inhibit the inflammatory pathway through decreased macrophage and neutrophil mural infiltration and decreased MMP2, 3, and 9 expression. Furthermore, IL-1B deletion is associated with preservation of elastin fibers and inhibition of smooth muscle cell phenotypic switching. IL-1B seems to play a central role in AAA formation and may illustrate future pharmacologic targets for AAA prevention.
Abstract 104: Apolipoprotein D: Implications for Multiple Roles in Lipid Metabolism
Authors: Srivastava Ujala , Abo-Ali Ehab M , Nguy Susanna , Lebegue Jean , Ali Kamilah
Published: July 2021

Introduction-

ApoD is a ubiquitously expressed protein that binds small hydrophobic ligands and is a minor component of lipoproteins. Polymorphisms of the human ApoD gene are associated with lipid abnormalities, specifically the reduction of HDL and ApoA1 levels. In fact, hepatic overexpression of ApoD has been shown to regulate the amount of plasma triglycerides. ApoD is also upregulated in human and mouse models of atherosclerosis, and is localized in cell types involved in atherosclerotic lesion formation. These data suggest that ApoD plays a role in lipid metabolism by modulating cellular processes in vascular cells during atherogenesis. In this study, our objective is to identify the role(s) of ApoD in lipid metabolism and to elucidate the mechanisms involved in this process.

Methods and Results-

To accomplish our objective, we used a two-pronged approach. We first studied the effect of ApoD on lipid metabolism on a chow diet. There was no significant difference between the levels of plasma cholesterol in ApoD -/- and wild-type mice on a chow diet; however, hepatic cholesterol levels had more than doubled. A 96-gene PCR array was used to assess differential expression of genes involved in fatty liver biogenesis. There was at least a 2-fold difference in expression in about 10 genes involved in insulin/glucose signaling, lipogenesis, and inflammation in the ApoD -/- mice. We then studied the effect of a Western diet in the ApoD -/- mice, which showed a significant reduction in plasma LDL-cholesterol and HDL-cholesterol when compared to wild-type mice. Analysis of the HDL fractions after subjecting plasma to a Fast Protein Liquid Chromatography column revealed increased levels of ApoA1 and Lecithin Cholesterol Acyl Transferase (LCAT) activity in ApoD -/- mice.

Conclusion-

A decrease in plasma cholesterol and an increase in ApoA1 and LCAT activity suggest that ApoD may play a role in the catabolism of HDL particles, resulting in lower plasma cholesterol levels in ApoD -/- mice. Our current data implies that ApoD plays a multifunctional role in lipid metabolism and the mechanism by which this occurs must be further examined.
Abstract 111: A Novel Assay that Incorporates Measurement of Ex Vivo LCAT Activity with Measurement of Cholesterol Efflux Capacity - Assessment of Two Biomarkers of Serum HDL Function
Authors: Collins Heidi L , Sulpizio Anthony C , Unruh Seanna M , Adelman Steven J
Published: July 2021

Atheroprotective properties of high-density lipoproteins (HDL) may be due to the ability to remove lipids from peripheral cells to the liver for excretion during reverse cholesterol transport (RCT). Assays that measure functionality of serum HDL are needed to evaluate the therapeutic potential of treatments targeting HDL metabolism. Recent work shows an inverse correlation between cholesterol efflux capacity of serum HDL and coronary artery disease, demonstrating the value of measuring HDL functionality. Another function of HDL that promotes RCT is the capacity for esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT). A novel assay was developed that in addition to measurement of serum HDL efflux capacity incorporates measuring the capacity for esterification of HDL-cholesterol by LCAT. Assay methodology includes measurement of global cholesterol efflux via all known pathways (ABCA1, SR-BI, ABCG1 and passive diffusion) from J774 macrophage cells to serum HDL, and measurement of the proportion of cholesterol transferred to serum HDL that is esterified by LCAT over 4h. Assay validation included assessment of intra-assay repeatability, inter-assay precision, linearity, LOQ and specificity. Results showed the intra-assay variability (%CV) from six independent preparations of serum HDL is ≤10% for both the % efflux and the % esterification of cholesterol. Inter-assay variability between four separate assays is ≤13% for both the % efflux and the % esterification of cholesterol. Cholesterol efflux is linear up to concentrations of 2.8% serum HDL and cholesterol esterification is linear up to concentrations of 4% serum. The LOQ for the assay is 0.15% cholesterol efflux and 0.40% cholesterol esterification. Global cholesterol efflux was determined to be specific for known cholesterol acceptors such as serum HDL (p≤0.0001 versus media blank). The cholesterol esterification measured was determined to be specific for the action of LCAT because the presence of LCAT inhibitor decreased the % ester by 86-97% (p≤0.0009). This novel assay allows the measurement of two functional aspects of an individual subject’s serum HDL, the capacities for both cholesterol efflux and for esterification of cholesterol by LCAT.
Abstract 116: A Targeted Sequencing Approach in Subjects with Extremely High HDL-C Identifies individuals with Potentially Causal Mutations in Both Alleles of 53 Genes
Authors: Larach Daniel B , Patel Aniruddh P , Peloso Gina M , Zanoni Paolo , DerOhannessian Stephanie L , Cuchel Marina , Kathiresan Sekar , Rader Daniel J
Published: July 2021

Introduction

The molecular etiology of extremely elevated HDL-C is poorly understood. Genome-wide association studies (GWAS) have identified many genetic loci associated with HDL-C, but very few of these harbor genes in which rare mutations are known to cause extremely elevated HDL-C levels. We performed targeted sequencing of exons in genes at GWAS lipid loci on subjects with extremely high HDL-C compared with those with low HDL-C in order to identify individuals with nonsynonymous mutations that could be causal for the extreme high HDL-C phenotype.

Methods

389 cases with HDL-C >95
th
percentile (102±19 mg/dL), and 387 controls with HDL-C <25
th
percentile (32±5 mg/dL) underwent targeted sequencing of exons in genes ±300 kb around each of 95 recently identified GWAS loci associated with lipid parameters. Candidate variants were identified by the following criteria: (1) missense, nonsense, and frameshift variants; (2) predicted to be deleterious by at least 1 of 3
in silico
prediction methods; (3) a ratio of high HDL-C heterozygotes to low HDL-C heterozygotes >1.5:1; (4) absence of any low HDL-C homozygotes; (5) MAF <0.01. Individuals with a potentially causal low-frequency variant in both alleles of a specific gene were identified for further phenotypic characterization.

Results

46 individuals with extremely high HDL-C were identified to have potentially causal low-frequency variants in both alleles of the same gene. These individuals were homozygous or compound heterozygous for mutations in 53 distinct genes. Most of these genes were not known to play defined roles in HDL metabolism, and include ARHGAP1, BTN2A2, CAMK2B, LILRA3, and OSCAR. These individuals are currently being called back for detailed phenotyping.

Conclusions

A targeted sequencing approach focused on genes at GWAS HDL-C loci in subjects with extremely high HDL-C identified 46 individuals with potentially causal low-frequency variants in both alleles of the same gene, including genes not known to play a role in HDL metabolism. More detailed phenotyping and family studies are underway, and are likely to provide new information regarding the phenotypic consequences of loss-of-function mutations in both alleles of several genes that influence HDL metabolism.
Abstract 123: Reciprocal Regulation of Plasma PCSK9 and Cell-surface Low-density Lipoprotein Receptor
Authors: Tavori Hagai , Fan Daping , Linton MacRae F , Fazio Sergio
Published: July 2021

Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. On the other hand, LDLR binds to and clears PCSK9 from the circulation, thus modulating its serum levels. To study the global and reciprocal effects of PCSK9 and LDLR on serum cholesterol, we developed transgenic mice expressing human (h) PCSK9 and characterized its activity, serum levels, and tissue distribution. Serum hPCSK9 concentration in transgenic mice was 2181±423 ng/ml, about 10 times higher than normal level in human serum. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation activity was most evident in the liver and small intestine. In contrast, LDLR levels were not affected by hPCSK9 expression in the adrenals and large intestine. On a chow diet, hPCSK9 transgenic mice on either C57BL/6 (wild-type, WT) or LDLR
-/-
background had higher cholesterol levels than their non-transgenic counterparts. Human PCSK9 transgenic mice had over a 4-fold increase in murine (m) PCSK9 serum levels compared to WT controls. However, transgenic expression of hPCSK9 in LDLR
-/-
mice did not affect the already elevated levels of mPCSK9. On the other hand, induction of hLDLR expression in transgenic mice caused a dramatic decrease in mPCSK9 levels. In addition hPCSK9 levels were increased by 2 fold in transgenic mice under LDLR
-/-
compare to WT background. Turnover studies with native PCSK9 showed rapid serum clearance in WT mice (half-life 5.2 min), whereas clearance was much slower in LDLR
-/-
recipient mice (50.5 min), and faster in hLDLR transgenic mice (2.9 min). In WT mice the injected PCSK9 accumulated in the liver and kidney but not in the adrenal gland. Ultracentrifugation and FLPC analysis showed that approximately one quarter of circulating hPCSK9 is associated with LDL, and that the LDL-associated PCSK9 is mainly in monomeric form. Our results show a reciprocal regulation between LDLR and PCSK9, which determines serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels. Understanding these interactions will increase our knowledge of serum cholesterol homeostasis and should provide the basis for an intelligent design of anti-PCSK9 therapies.
Abstract 135: Exploration and Modulation of TREM-1 in Experimental Atherosclerosis
Authors: Potteaux Stephane , Joffre Jeremie , Esposito Bruno , Tedgui Alain , Gibot Sebastien , Mallat Ziad , Ait Oufella Hafid
Published: July 2021

Under conditions of atherosclerosis, monocytes are rapidely recruited into the vessel wall where they differentiate into macrophages. Both classical and nonclassical monocytes are continuously recruited to the lesion and contribute to atherosclerosis formation. Whereas the number of circulating monocytes correlates with plaque development, decrease in their number or migration is protective.

Upon differentiation, macrophages increase TLR expression, which triggers the inflammatory response.

The triggering receptor expressed on myeloid cells (TREM-1) has been shown to amplify TLR-induced signals in sepsis or inflammatory bowel disease. TREM-1 is also produced in a soluble form and is a predictive factor during sever infections in humans. We addressed for the first time the role of TREM-1 in atherosclerosis.

We found preferential expression of TREM-1 on blood neutrophils and nonclassical monocytes in both ApoE+/+ and ApoE-/- mice, but sTREM-1 was only detectable in plasma of ApoE-/- mice (ELISA). TREM-1 expression was significantly increased on both cell types in APOE-/- mice after 4 weeks of high fat diet (Flow cytometry). We next addressed the role of TREM-1 in atherosclerosis formation by injection of a TREM-like transcript 1-derived peptide (LR12) in 8 week-old ApoE-/- mice (daily injection for 4 weeks of LR12 or peptide scramble as control).

We found that pharmaceutical inhibition of TREM-1 significantly reduced plaque formation by 30% without change in cholesterol levels. This was associated with significant reduction in macrophage accumulation after treatment with LR12 (57735,34 μm2 vs 78398,38 μm2 in controls; p=0,004). We demonstrated that LR12 treatment induced a specific rapid and sustained decrease in circulating nonclassical monocytes after 7 days of treatment. By using an in vivo pulse labeling method to quantify monocyte migration, we found that LR12 also altered nonclassical monocyte recruitment to the plaque.

Taken together, these data indicate that TREM-1 expression increases in the context of atherosclerosis and that pharmacological inhibition of TREM-1 protects against plaque development through decreased number and infiltration of nonclassical monocytes.
Abstract 140: Anti-inflammatory and Anti-atherogenic Role of Bmp Receptor II In Endothelial Cells
Authors: Kim Chanwoo , Song Hannah , Kumar Sandeep , Nam Douglas , Kwon Hyuk Sang , Chang Kyung Hwa , Son Dong Ju , Kang Dong-Won , Brodie Seth A , Weiss Daiana , Vega J. David , Albert-Grill Noah , Griendling Kathy , Taylor W. Robert , Jo Hanjoong
Published: July 2021

Atherosclerosis is a multifactorial disease that arises from a combination of endothelial dysfunction and inflammation, occurring preferentially in arterial regions exposed to disturbed flow. Bone morphogenic protein-4 (BMP4) produced by disturbed flow induces inflammation, endothelial dysfunction and hypertension, suggesting the importance of BMPs in vascular biology and disease. BMPs bind to two different types of BMP receptors (BMPRI and II) to instigate intracellular signaling. Increasing evidences suggest a correlative role of BMP4 and atherosclerosis, but the role of BMP receptors especially BMPRII in atherosclerosis is still unclear and whether knockdown of BMPRII is the cause or the consequence of atherosclerosis is still not known. It is therefore, imperative to investigate the mechanisms by which BMPRII expression is modulated and its ramifications in atherosclerosis. Initially, we expected that knockdown of BMPRII will result in loss of pro-atherogenic BMP4 signaling and will thereby prevent atherosclerosis. Contrarily, we found that loss of BMPRII expression causes endothelial inflammation and atherosclerosis. Using BMPRII siRNA and BMPRII
+/-
mice, we found that BMPRII knockdown induces endothelial inflammation in a BMP-independent manner via mechanisms involving reactive oxygen species (ROS), NFκB, and NADPH oxidases. Further, BMPRII
+/-
ApoE
-/-
mice develop accelerated atherosclerosis compared to BMPRII
+/+
ApoE
-/-
mice, suggesting loss of BMPRII may induce atherosclerosis. Interestingly, we found that multiple pro-atherogenic stimuli such as hypercholesterolemia, disturbed flow, pro-hypertensive angiotensin II, and pro-inflammatory cytokine, TNFα, downregulate BMPRII expression in endothelium, while anti-atherogenic stimuli such as stable flow and statin treatment upregulate its expression, both
in vivo
and
in vitro
. Moreover, we found that BMPRII expression is significantly diminished in human coronary advanced atherosclerotic lesions. These results suggest that BMPRII is a critical, anti-inflammatory and anti-atherogenic protein that is commonly targeted by multiple pro- and anti-atherogenic factors. BMPRII could be used as a novel diagnostic and therapeutic target in atherosclerosis.
Abstract 141: The Loss of Nrf2 Enhances Vascular Inflammation but Reduces Atherosclerosis via Effects on Plasma Lipids in LDL Receptor Deficient Mice Fed High Fat Diet
Authors: Ruotsalainen Anna-Kaisa , Tapper Emmi , Lappalainen Jari , Ylä-Herttuala Seppo , Levonen Anna-Liisa
Published: July 2021

OBJECTIVE

Transcription factor NF-E2-related factor 2 (Nrf2) regulates antioxidant and detoxifying enzymes affording cytoprotection in the cardiovascular system. We have previously reported that Nrf2 deficiency specific to bone marrow derived cells aggravates atherosclerosis in LDL receptor deficient (LDLR-/-) mice. Furthermore, Nrf2 deficiency in macrophages enhances foam cell formation and promotes the proinflammatory phenotype. In contrast, the total loss of Nrf2 has been shown to protect against atherogenesis in apoE-deficient mice. The mechanism by which Nrf2 deficiency affords atheroprotection in apoE-model is currently unknown, but combined systemic and local vascular effects have been proposed. Given these contrasting results, we aimed at assessing the effect of total loss of Nrf2 on atherogenesis in hypercholesterolemic LDLR-/- mice.

METHODS AND RESULTS

Nrf2-/- mice were cross-bred to LDLR-/- mice and fed a high fat diet (HFD) for 6 or 12 weeks. The degree of atherosclerosis was assessed from the cross-sections of proximal aorta. Nrf2 deficiency decreased atherosclerosis in females from 17.2±8.7 % (mean ±SD, n=11-25) to 12.3±4.6 % (p=0.1) and in males from 11.2±3.9 % to 7.6±4.6 % (p=0.025) after 6 weeks on HFD. After 12 weeks on HFD the effects were more pronounced, as Nrf2 deficiency decreased atherosclerosis from 36.5±5.5 % to 30.3±3.5 % in females (p=0.001) and from 30.6±5.7 % to 21.3±6.9 % in males (p=0.005). Nrf2 deficiency also increased the macrophage content relative to lesion area. Supporting systemic effects, Nrf2 deficiency reduced plasma total cholesterol from 24.0±8.4 mmol/l to 10.6±6,7 mmol/l (p=0.006) and triglyceride levels from 3.6±1.4 mmol/l to 1.7±1.2 mmol/l (p=0.008) in males after 6 weeks on HFD.

CONCLUSIONS

In contrast to bone marrow specific Nrf2 deficiency, total loss of Nrf2 aggravates atherosclerosis in LDLR-/- mice likely via systemic effects on lipid metabolism.
Abstract 168: Impact of Obesity and Atrial Fibrillation on Arterial Stiffness
Authors: Willoughby Scott R , Dickinson Kacie , Schultz Carlee , Sanders Prashanthan , Clifton Peter , Keogh Jennifer
Published: July 2021

Introduction

Turbulent blood flow which occurs in atrial fibrillation patients due to an irregular heart rate may lead to vascular abnormalities and increased thrombotic risk. Obesity is an important emerging substrate for the development of atrial fibrillation. Obesity is also associated with endothelial dysfunction and increased arterial stiffness. We sought to determine the impact of obesity on arterial stiffness in patients with atrial fibrillation.

Methods

34 patients with atrial fibrillation (age 59±12 years,) were evaluated in lean (n=21) and obese (n=13) subgroups, and compared to age-matched lean (n=15) and obese (n=14) control subjects. Arterial stiffness was assessed using radial applanation tonometry (SphygmoCor). Heart rate adjusted aortic augmentation index (AIx: a measure of arterial stiffness) was calculated from the corresponding waveforms utilizing validated transfer functions.

Results

Obese controls and obese atrial fibrillation patients had significantly higher AIx (arterial stiffness) than lean controls and lean atrial fibrillation patients (p=0.003 and p=0.03, respectively: Figure). There was no difference in AIx scores between obese atrial fibrillation patients and obese control subjects (p=0.3) and between lean atrial fibrillation and lean control subjects (p=0.8).

Conclusion

We conclude that obesity is associated with arterial stiffness and atrial fibrillation does not add additional burden. These results suggest the interaction between obesity and atrial fibrillation may contribute to the observed increase in thromboembolic risk, in part, through vascular abnormalities other than increased arterial stiffness.
Abstract 169: Type 2 Diabetes-Induced Hematopoietic Stem Cell Oxidant Stress Attenuates the Differentiation, Skews M1/M2 Specification of Monocytes/Macrophages and Delays Wound Healing in db/db Mice
Authors: Yan Jinglian , Tie Guodong , Wang Shouying , Messina Louis
Published: July 2021

Rationale

After recruitment to wounds, monocytes differentiate into macrophages which play a central role in all stages of wound healing. Wound healing is significantly delayed in type 2 diabetes and it is related to macrophage specification into M1/M2 phenotype, but the mechanism remains unknown.

Objective

This study tested the hypothesis that type 2 diabetes induces hematopoietic stem cells (HSCs) oxidant stress that reduces their differentiation towards monocytes and skews the specification of M1/M2 phenotype, thereby causing delayed wound healing.

Methods and Results

HSCs were sorted from bone marrow of WT and db/db type 2 diabetic mice. DCF staining showed significant oxidant accumulation in HSCs from db/db mice which was reversed by the antioxidant, N-acetylcysteine (NAC). Bone marrow monocyte concentration (FACS analysis of cell surface markers f4/80, cd14 and cd115) was significantly lower in db/db mice than in WT mice. NAC also reversed the reduced differentiation towards monocyte. Wound closure rate was significantly delayed in db/db mice. Macrophages were isolated from wounds and their concentration and M1/M2 phenotype were quantified by flow cytometry. During the
inflammatory phase
of wound healing, macrophage concentration was decreased and the proportion of M1 macrophage was lower in db/db mice than in WT mice. During
new tissue formation phase
, macrophage concentration was decreased and the proportion of M2 macrophage was lower, but M1 macrophage was higher in db/db mice than in WT mice. During
tissue remodeling phase
, macrophage concentration was increased and M1 macrophage remained higher in db/db mice, but no difference was observed in the proportion of M2 macrophage. The reduced differentiation of HSCs towards monocytes and the delayed wound closure phenotype of db/db mice could be transferred to WT mice by transplanting db/db HSCs into lethally irradiated WT mice.

Conclusion

Type 2 diabetes-induced HSC oxidant stress impairs HSC differentiation towards monocytes, skews the M1/M2 specification of macrophages and thereby accounts for the delayed wound healing. Type 2 diabetes-induced HSC oxidant stress may be a heretofore unrecognized critical regulator of dysinflammation in type 2 diabetes.
Abstract 172: The Effects of Renal Function on Platelet Reactivity in Patients Treated with Clopidogrel and Admitted for Major Adverse Cardiovascular Events in a Community Setting
Authors: Davila Carlos , Vargas Fernando , Monaco Thomas , Huang Kuan-Hsiang Gary , Morris D L , Figueredo Vincent M , Christian Witzke
Published: July 2021

Introduction

Increased platelet reactivity (PR) while on antiplatelet therapy is associated with worse outcomes. There are conflicting results regarding the influence of renal function on PR in patients taking clopidogrel. Here we assessed the relationship between renal function, PR, and outcomes in a community practice setting.

Methods

We retrospectively reviewed 98 consecutive, non-dialysis patients admitted for major adverse cardiovascular events (MACE) to our institution between 2011 and 2012 that had PR values measured with the VerifyNow P2Y12 Assay. High PR was defined as a PRU >230 and low PR as a PRU ≤178. Renal function was classified based on estimated glomerular filtration rate at the time of assay. All cause mortality, readmissions, length of stay (LOS), and number of cardiac catheterizations were reviewed over 455 days.

Results

The prevalence of PRU>230 was 100% in stage 4 and 5 CKD, 42.9% in stage 3, 53.8% in stage 2, and 53.3% in stage 1 (p=0.049, figure 1). In patients presenting with acute coronary syndromes (n=49), a significant positive association existed between PRU and total number of cardiac catheterizations (p=0.014). Overall patients with PRU value >230 had longer LOS when compared to those with PRU≤230 (median of 2 days vs. 1 day, p=0.049). A positive correlation between HbA1c and PRU was also found (p=0.018). All 8 deaths had abnormal PRU (3 patients with PRU≤178, 5 patients with PRU>230).

Conclusion

Advanced CKD is associated with impairment of platelet inhibition by clopidogrel measured with the VerifyNow P2Y12 Assay. Elevated PRU is associated with worse outcomes. We observed a significant correlation between HbA1c and degree of platelet inhibition.
Abstract 180: Differential Role of Endothelin-1 in Mitral Stenosis and Atrial Fibrillation
Authors: Willoughby Scott R , Schultz Carlee , Kumar Sathesh , Nair Sukesh , Srivastava Alok , Joseph George , Chandy Sunil , John Bobby , Sanders Prashanthan
Published: July 2021

Background

Chronic atrial stretch due to mitral stenosis is a major substrate for the development of atrial fibrillation (AF). It is well established that rhuematic mitral stenosis and AF are associated with an increased risk of thromboembolic events. Endothelin-1 (ET-1) is a potent vasoconstrictor and modulator of vascular function, and is known to play a role in thrombus formation. We therefore sought to compare ET-1 levels between patients with rheumatic mitral stenosis and AF.

Methods

We measured plasma levels of ET-1 in blood samples from the femoral vein (FV), right atria (RA) and left atria (LA) obtained from 19 patients with rheumatic mitral stenosis, 12 patients with lone AF and 10 subjects with supraventricular tachycardia (reference subjects [REF]). Plasma ET-1 levels were measured by conventional ELISA techniques.

Results

The FV concentrations of ET-1 were significantly higher in rheumatic mitral stenosis patients than in AF patients or REF subjects (Figure). However, there was no significant difference in the concentrations of ET-1 between AF patients and REF subjects. In rheumatic mitral stenosis patients and REF subjects there were no differences in concentrations of ET-1 between the FV, RA and LA. In contrast, ET-1 levels were highest in the femoral vein of AF patients (FV vs RA p<0.03 and FV vs LA p<0.06).

Conclusion

Patients with rheumatic mitral stenosis have significantly higher ET-1 levels than AF patients. These data suggest that ET-1 plays a differential role in the pathophysiology of thrombus formation in mitral stenosis and AF. It highlights the importance of the underlying substrate to the risk of developing thrombus within the atria.
Abstract 183: Differences in Genetic Regulation of Carotid Stenosis and Thickening in Mice
Authors: Gerloff Janice , Genyn Katrien , Lusis Aldons J , Korshunov Vyacheslav A
Published: July 2021

Introduction

Most clinical and experimental studies are focused on the artery wall using a carotid intima-media thickening (IMT) phenotype. In physiological remodeling increases in IMT are coupled with increased arterial dimensions that preserve arterial blood flow. In contrast, pathological remodeling results in arterial stenosis. The goal of this study was to investigate genetic mechanisms of the carotid stenosis in inbred mice.

Methods and Results

We ligated external and internal branches of the left carotid artery, leaving the occipital artery patent to induce carotid IMT in mice. We evaluated flow-induced carotid remodeling in 9-12 week old males of 30 inbred mouse strains (n=270). Relative reductions in the left carotid blood flow were very similar among the mouse strains (Mean+SD= -86+7%) after two weeks after ligation. All animals were perfusion fixed with formalin and carotid arteries were stained with hematoxylin and eosin. There was less than 2-fold variation in carotid traits in controls. However, ligation resulted in a greater variation of carotid IMT (~6-fold) compared to carotid stenosis (~3-fold). The highest carotid IMT values were in SEA/GnJ, while the lowest IMT was in C3H/HeJ mice. Carotid stenosis was >60% in SEA/GnJ and SJL/J strains. There was significant correlation (R
2
=0.40) between carotid stenosis and IMT across 30 strains. We performed genome-wide association by using Efficient Mixed Model Association method for carotid traits in 30 inbred mice. Comparable numbers of genetic loci were associated with carotid IMT and stenosis after ligation. However, none of these loci overlap between the carotid stenosis and carotid IMT.

Conclusions

We found that major genetic modifiers for carotid stenosis are different from those that control carotid IMT. Our data also suggest that identification of mechanisms that determine carotid stenosis is difficult through studies on the carotid IMT.
Abstract 186: Aortic IL-17C Producing Cells in Atherosclerosis.
Authors: Butcher Matthew J , Galkina Elena V
Published: July 2021

The progression of atherosclerosis from fatty acid streaks to complex lesions involves regular communication between hematopoetic cells within the arterial wall and vascular cells via cytokine and chemokine networks. Recent evidence has demonstrated the presence of Interleukin-17A (IL-17A)-producing T cells within atherosclerotic Apolipoprotein E-deficient (Apoe-/-) aortas. IL-17A is a member of the IL-17 cytokine family, which consists of six related isoforms (IL-17A-IL-17F). While the functions of IL-17A, IL-17E, and IL-17F are the subject of ongoing studies, the roles and producers of IL-17B and IL-17C remain elusive. Recent studies have demonstrated a pro-inflammatory role for epithelial cell and keratinocyte-derived IL-17C in the context of experimental colitis, and psoriasis. In the present study, we sought to examine the expression of IL-17C within the aortas of atherosclerotic Apoe-/- and Il17a-/-Apoe-/- mice. We detected Il17c mRNA within the aortas of both 12-week western diet-fed Apoe-/- and Il17a-/-Apoe-/- mice. To further identify and characterize the cellular sources of aortic IL-17C, we examined aortic leukocytes and vascular cells by flow cytometry. We identified several major cellular producers of IL-17C in vivo, including a major population of smooth muscle cells (5±0.8% of Apoe-/- CD45-αSMA+ SMCs) and a population of CD45-αSMA- cells (5.8±0.8% of Apoe-/- CD45-αSMA- cells). Interestingly, the percentage of aortic IL-17C producing cells was elevated within 12 week western diet-fed Il17a-/-Apoe-/- mice in comparison to Apoe-/- littermates (6.16±0.24% of the Il17a-/-Apoe-/- aorta vs 3.6±0.1% of the Apoe-/- aorta), suggesting an inverse relationship between T cell derived-IL-17A and vascular-IL-17C. To examine the effects of IL-17C and the possibility of synergy between TNFα and IL-17C on aortic chemokine and cytokine production, we explanted and treated whole aortas with IL-17C or IL-17C and TNFα in vitro. IL-17C with and without TNFα broadly supported the expression of Ccl2, Ccl7, Cxcl1, Cxcl2, Cxcl5, and Il6 in explanted WD Apoe-/- aortas. Together, these results suggest that vascular smooth muscle cell-derived IL-17C may play a pro-inflammatory role in atherosclerosis via the production of chemokines and cytokines.
Abstract 188: Immobilization Stress Exacerbates Insulin Resistance and Endothelial Dysfunction via Inhibition of Insulin-Stimulated eNOS Activation and via Enhancement of NF-kB Activation by Corticotropin-Releasing Hormone
Authors: Chung Ick-Mo , Kim Hee Young , Lee Youn Jung , Lee Kuy-Sook , Shin Mi-Kyung
Published: July 2021

Objectives

Psychological stress may contribute to the development of atherosclerosis and insulin resistance. However, its mechanism is poorly understood. We studied if stress induces insulin resistance and endothelial dysfunction, and assessed the role of stress-associated corticotropin-releasing hormone (CRH) in inflammation and insulin signaling pathways.

Methods and Results

Immobilization stress (IS) was applied (2 hr/d) for 14 d to male 8-wk (YI2O: before DM phenotype) and 21-wk (EI2O: DM phenotype) OLETF rats, whereas control groups comprised of rats without IS with same age (YCO and ECO, respectively; n=7~10 for each group). Systolic blood pressure was significantly increased after 14d-IS in YI2O and EI2O, whereas it was not changed in control groups. Acetylcholine-induced, but not nitroprusside-induced, vasorelaxation, measured in superior mesenteric artery, was significantly decreased by 14d IS compared with each control groups, suggesting that IS induced endothelial dysfunction. 14d-IS increased plasma insulin (342.5±6.3 vs 496±51.6 pg/ml) and HOMA-IR (3.1±0.2 vs 4.7±0.5 mmol/LxμIU/mL) in 8-wk OLETF rats (control vs stress, all
p
<0.05). Western blot analysis using thoracic aorta showed 14d-IS increased expression of Rho-associated kinase-1 and p22
phox
subunit of NAD(P)H oxidase compared with control groups. eNOS phosphorylation at Ser
1177
was increased by treatment with insulin (2nM) in cultured HUVEC cells, but this was blocked by pretreatment with CRH (100 nM). On the contrary, an increase in ERK phosphorylation by insulin treatment was further enhanced by CRH pretreatment in HUVEC cells. CRH also induced translocation of NF-kB subunit p65 into nuclear fraction in monocyte cell line U937 cells.

Conclusions

Immobilization stress exacerbates insulin resistance and endothelial dysfunction in a prediabetic animal model at least partly via inhibition of insulin-mediated eNOS activation and via enhancement of inflammation through ERK pathway and NF-kB activation by CRH.
Abstract 194: Increased Monocyte Adhesion and Infiltrative Activity Inhibits Arteriovenous Fistula Maturation for Hemodialysis Access in Patients With End-stage Renal Disease
Authors: Samadzadeh Kiana M , Van Spyk Elyse N , Chun Kevin C , Lee Eugene S
Published: July 2021

Introduction

Patients with end-stage renal disease (ESRD) require hemodialysis access and arteriovenous fistulas (AVFs) are the preferred method for hemodialysis access. However, 60% of surgically constructed AVFs do not functionally mature and 80% of the reported AVF failures are caused by stenosis at the venous outflow of the fistula. Stenosis is commonly associated with deregulated inflammatory mechanisms and prolonged systemic inflammation is proposed to upregulate monocyte activity within the vessel, affecting vascular remodeling.

Hypothesis

We hypothesize that excessive monocyte interaction with the vessel endothelium from systemic inflammation attenuates AVF maturation and prevents appropriate vascular remodeling. The purpose of this study is to compare monocyte activity through adhesion molecule expression, adhesion to endothelium, and transendothelial migration in patients with a matured AVF versus patients with a failed AVF.

Methods

Peripheral blood was collected from control subjects (n=4) and ESRD patients with failed (n=4) and matured (n=8) AVFs. Endothelial cell adhesion, transendothelial migration, and permeability assays were assessed under static conditions. Flow cytometry was used to determine the presence of monocyte adhesion molecules CD18, CD11a, CD11b, and CD11c.

Results

Monocytes from failed AVF patients exhibited greater expression of CD18 (p= 0.03) and CD11b (p= 0.03) compared to matured AVF patients. Monocytes from failed AVF patients had greater adherence to endothelium in untreated (p= 0.02) conditions compared to matured AVF patients. Monocytes from failed AVF patients had insignificantly greater transendothelial migration towards fMLP (p= 0.2) compared to matured AVF patients. Monocytes from all ESRD patients had more adhesion to endothelium compared to controls (p= 0.01). All ESRD patients had greater transmigration towards fMLP (p= 0.03) compared to controls.

Conclusion

In conclusion, monocytes isolated from patients with failed AVFs have greater CD18/CD11 expression, cell adhesion to the vessel endothelium, and endothelial dysfunction than those from matured AVFs.
Abstract 199: Anticoagulant Heparan Sulfate Conveys Anti-Inflammatory Activity of Antithrombin
Authors: Smits Nicole C , Shworak Nicholas W
Published: July 2021

Antithrombin (AT) therapy reduces morbidity/mortality in systemic inflammatory response syndromes, such as septic shock. These effects stem from the ability of AT to directly initiate cell signaling, which requires AT engagement with heparan sulfate proteoglycans. It is presently unclear if this process involves anticoagulant heparan sulfate (HS
AT+
), a specific motif with high affinity for AT that is produced by endothelial cells. If HS
AT+
regulates anti-inflammatory signaling, then

Hs3st1
-/-

mice, which lack endothelial HS
AT+
, should exhibit a proinflammatory state and should not benefit from AT treatment. We tested these hypotheses using a mouse model of lipopolysaccharide (LPS)-induced septic shock. Hemodynamics were monitored with a left ventricle pressure-volume catheter. Both
Hs3st1
+/+
and
Hs3st1
-/-
mice die of cardiogenic shock. However,
Hs3st1
-/-
, compared to
Hs3st1
+/+
, mice exhibited a much more rapid onset of death. Left ventricular gene expression profiles of mice with systemic LPS challenge showed enhanced expression of leukocyte recruitment genes (P-selectin, Cxcl10) in
Hs3st1
-/-
mice. Consistent with these data, LPS-treated
Hs3st1
-/-
mice, compared to
Hs3st1
+/+
, exhibited enhanced leukocyte rolling flux within cremaster muscle venules. Thus, mice lacking HS
AT+
exhibit a proinflammatory phenotype. AT treatment of
Hs3st1
+/+
mice reduced both LPS-induced lethality and leukocyte-endothelial interactions. Conversely AT treatment of
Hs3st1
-/-
mice enhanced both LPS-induced lethality and leukocyte-endothelial interactions. Thus, AT failed to elicit anti-inflammatory effects in mice lacking HS
AT+
. Our results indicate that endothelial HS
AT+
is one component that mediates AT’s anti-inflammatory activity. These findings provide an initial step towards elucidating the role of HS
AT+
and AT in a novel natural anti-inflammatory pathway of the blood vessel wall. This pathway is an ideal target for developing therapeutics to combat the devastating outcomes of septic shock.
Abstract 2: Overexpression of Human Apolipoprotein C-III in Mice Decreases Intestinal Transport of Triglyceride Into Lymph
Authors: Kohan Alison B , Wang Fei , Yang Qing , Huesman Sarah , Dong H. H , Hui David Y , Tso Patrick
Published: July 2021

INTRODUCTION

Apolipoprotein C-III (apoC-III), synthesized by the liver and intestine, is an inhibitor of LPL-mediated lipolysis and hepatic clearance of triglyceride-rich lipoproteins. ApoC-III overproduction is linked with hypertriglyceridemia and atherosclerosis. ApoC-III may also play an intracellular role in hepatic VLDL assembly/secretion. Little is known about the role of apoC-III in the intestine, although it is secreted on chylomicrons coincident with triglyceride absorption.

HYPOTHESIS

Given that overexpression of apoC-III results in high plasma triglyceride levels, we hypothesized that it might also stimulate intestinal triglyceride transport, thereby exacerbating plasma hypertriglyceridemia in human apoC-III transgenic (h-apoC-III tg) mice.

METHODS

28-30 gram male h-apoC-III tg (on a C57BL/6J background) were fitted with both a mesenteric lymph cannula and a duodenal feeding tube, and received a continuous intraduodenal infusion of triglyceride (6 μmol of 3[H]-Triolein in 0.3ml of phosphate-buffered saline) for 6 hours with hourly lymph samples collected (n=11-12). At the end of the infusion period, luminal and mucosal contents and tissue samples were isolated. An advantage of this lymph fistula model is the ability to sample lymph continuously throughout the triglyceride infusion period while avoiding confounding effects of anesthesia and stomach emptying.

RESULTS

h-apoC-III tg mice had a decrease in lymph flow and a 43% reduction in lymphatic 3[H]-triglyceride transport compared to WT mice. The h-apoC-III tg mice had 10.0±2.3% of the total dose of 3[H]-triglyceride remaining in intestinal lumen; which was significantly higher than the 3.1±0.4% observed in WT mice. Thin layer chromatographic analysis of the luminal contents showed that h-apoC-III tg mice, as opposed to WT controls, had a significantly higher percentage of fatty acid. There were no significant differences in the luminal triglyceride, diglyceride, and monoglyceride composition between groups.

CONCLUSION

Our studies reveal a novel role for apoC-III in decreasing intestinal triglyceride transport distinct from its extracellular roles in plasma on lipoprotein lipase, and its intracellular role in hepatic VLDL synthesis and secretion.