bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 86: Intercellular Adhesion Molecule 1 Deficiency Attenuated Angiotensin II--Induced Abdominal Aortic Aneurysms in Apolipoprotein E--Deficient Mice
Authors: Uchida Haruhito A , Umebayashi Ryoko , Kakio Yuki , Shikata Kenichi , Makino Hirofumi
Published: July 2021

Objective:
Chronic infusion of angiotensin II (AngII) augments the development of abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Several studies have suggested that intercellular adhesion molecule 1 (ICAM-1) expression increases in association with AAA formation. The aim of the study was to define whether ICAM deficiency influenced AngII-induced AAA formation.

Methods and Results:
Apolipoprotein E deficient (apoE -/-) mice were cross-bred with ICAM-1 deficient mice. Male apoE -/- mice fed a normal diet and infused subcutaneously with saline or AngII (1,000 ng/kg/min) via osmotic mini pump for 1 week. AngII infusion increased aortic ICAM-1 protein. Male apoE -/- mice that were either ICAM-1 +/+ or -/- were fed a normal diet and infused subcutaneously with AngII (1,000 ng/kg/min) via osmotic mini pump for 4 weeks. Total ICAM-1 deficiency had no significant effect on body weight, total cholesterol concentrations, or systolic blood pressures prior to and during AngII infusion. AngII induced expansion of ex vivo maximal diameters of abdominal aortas was attenuated significantly in ICAM-1 deficient mice (ICAM-1 +/+, 1.78 ± 0.20 mm; ICAM-1 -/-, 1.07 ± 0.03 mm, P < 0.0001). ICAM-1 deficiency also reduced significantly the incidence of AngII-induced AAAs (ICAM-1 +/+, 76%; ICAM-1 -/-, 13%, P < 0.0001). Furthermore, bone-marrow transplantation was performed to develop chimeric mice that were ICAM-1 +/+ or -/- in donor cells and ICAM-1 +/+ or -/- in recipient cells. ICAM-1 deficiency in either donor or recipient cells had no effect on body weight, total cholesterol concentrations, or systolic blood pressure. Recipient ICAM-1 deficiency significantly attenuated the incidence of AngII-induced AAA formation (ICAM-1 +/+, 67%; ICAM-1 -/-, 19%, P = 0.0008). Furthermore, lack of ICAM-1 reduced AngII-induced aortic MMP-2 activation (P < 0.05).

Conclusion:
ICAM-1 deficiency attenuated AngII-induced AAAs in male apoE -/- mice.
Abstract 114: Serum Amyloid A Plays an Important Role in Making HDL Lose Its Anti-inflammatory Effect on Adipocytes
Authors: Han Chang Yeop , Guevara Myriam E , Wei Hao , Tang Chongren , Omer Mohamed , Wang Shari , de Beer Maria C , de Beer Frederick C , Osborne William R , Elkon Keith B , Chait Alan
Published: July 2021

Adipose tissue inflammation associates with insulin resistance and increased cardiovascular disease risk. HDL has both anti-inflammatory and atheroprotective properties. We recently showed that HDL from healthy humans and lean mice inhibits palmitate-induced monocyte chemotactic factor expression by decreasing lipid raft (LR) content in adipocytes. However, little is known regarding whether inflammation would cause HDL to lose these anti-inflammatory effects on adipocytes. To generate HDL under conditions of sterile inflammation, HDL was purified from plasma of AgNO
3
-injected C57BL/6 mice. In 3T3L-1 adipocytes, “inflamed HDL” did not disrupt the formation of LR or inhibit monocyte chemotactic factor expression. Moreover, inflamed HDL lost its ability to facilitate cholesterol efflux in adipocytes. To determine causal factors of HDL that might be responsible for this loss of anti-inflammatory effect on adipocytes, we investigated role of serum amyloid A (SAA), levels of which are increased up to 1000 fold in HDL by acute inflammation. In a loss of function study, inflamed HDL from AgNO
3
-injectd SAA1/2 double knockout (DKO) mice restored part of its anti-inflammatory and cholesterol efflux capacity. Conversely, in a gain of function study, increasing the SAA content in uninflamed HDL by exposure to 293 HEK cells with lentiviral overexpression of SAA2 resulted in a partial loss of its anti-inflammatory effect and cholesterol efflux capacity. To determine whether HDL from moderately inflamed humans had similar effects on adipocyte inflammation, HDL isolated from patients with systemic lupus erythematosus (n=18) also demonstrated impaired anti-inflammatory effects in adipocytes. The extent of impairment associated positively with plasma SAA levels (
r
=0.80;
p
<0.001), and negatively with cholesterol efflux capacity of the HDL (
r
=-0.74;
p
<0.001). These findings indicate that inflamed HDL loses its anti-inflammatory effect and cholesterol efflux capacity on adipocytes. The presence of SAA in HDL may contribute to the generation of dysfunctional HDL and should be considered as a causal factor, not simply as a biomarker of dysfunctional HDL.
Abstract 129: Effects of a Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitor, Alirocumab, on Lipid and Lipoprotein Metabolism in Normal Subjects
Authors: Reyes-Soffer Gissette , Pavlyha Marianna , Ngai Colleen , Ippolito Paul , Holleran Stephen , Ramakrishnan Rajasekhar , Karmally Wahida , Donovan Daniel , Cowan Lisa , Sasiela William , Surks Howard , Rey Jacques , Ginsberg Henry
Published: July 2021

Background:
Proprotein convertase subtilisin/kexin type (PCSK9) inhibitors are promising new drugs for the treatment of hypercholesterolemia. They inhibit the binding of PCSK9 to the low density lipoprotein (LDL) receptor that, in turn, decreases lysosomal degradation of LDL receptors and increases their numbers on the cell surface. In Phase 2/3 studies, alirocumab significantly lowered plasma levels of LDL-cholesterol (C) and apolipoprotein B (apoB). The mechanism underlying the LDL-C lowering effects of PCSK9 inhibition has not been reported.

Method:
We enrolled 10 healthy volunteers (4 male, 6 female), into a Phase 1, placebo-controlled, single-blind, single-sequence study to examine the effects of alirocumab, 150 mg administered subcutaneously every two weeks, on lipid and lipoproteins levels and the metabolism of apoB in very low density (VLDL), intermediate density (IDL) and LDL. Subjects received 2 doses of placebo followed by 5 doses of alirocumab. At the end of each treatment period, fasting lipids and lipoprotein levels were measured, and stable isotope studies of the apoB turnover in VLDL, IDL and LDL were performed.

Results:
Alirocumab significantly reduced plasma levels of total-C by 37% (178.4±34 to 112.7±29 mg/dL), LDL-C by 59% (110.2±25 to 45.5±26 mg/dL), and apoB by 51% (93.6±25 to 45.5±13 mg/dL) compared to placebo. Plasma triglycerides (TG) and HDL-C did not change. Levels of LDL-C, LDL-TG, and LDL-apoB fell by 55.8±10%, 33.9±13%, and 56.0±11%, respectively (all p<0.0001), on alirocumab. The reductions in LDL apoB were explained by a dramatic increase in the fractional clearance rate (FCR) of LDL apoB from 0.50±0.18 on placebo to 1.02±0.35 pools/day on alirocumab (p<0.001) and a trend toward lower LDL apoB production rates on alirocumab (15.1±4.6 vs 12.9±3.3 mg/kg/day; p=0.10). Levels of IDL-C, IDL-TG and IDL-apoB were also reduced significantly and there was a trend toward an increase in IDL apoB FCR on alirocumab (placebo: 9.2±4 vs alirocumab: 10.8±3 pools/day; p=0.06). Additional kinetic parameters will be presented at the meeting.

Summary:
Alirocumab treatment significantly reduced the levels of IDL and LDL, and these changes were due to increases in the FCRs of these lipoproteins, particularly LDL.
Abstract 131: Restoration of ER Stress and Induction of FGF15/19 Independently Rescue ABCG5 ABCG8 Sterol Transporter
Authors: Wang Yuhuan , Su Kai , Sabeva Nadezhda , Ji Ailing , van der Westhuyzen Deneys , Foufelle Fabienne , Gao Xia , Graf Gregory
Published: July 2021

Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant and have reduced levels of biliary cholesterol due, in part, to reduced levels of hepatic ABCG5 ABCG8 (G5G8). Chronic leptin replacement restores G5G8 abundance and increases biliary cholesterol concentrations, but the molecular mechanism responsible for G5G8 regulation remains unclear.

In the current study, we conducted a series of experiments to address potential mechanisms. To determine if leptin signaling directly regulates hepatic G5G8 abundance, we acutely replaced leptin in ob/ob mice and deleted hepatic leptin receptors in lean mice. Neither manipulation altered G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy had no effect on G5G8.

Alternatively, the G5G8 protein complex may be decreased due to compromised ER stress. It has been previously reported that tauroursodeoxycholate (TUDCA) alleviates ER stress. It also increases G5G8 and biliary cholesterol in both lean and db/db mice. The ER chaperone protein, glucose regulated protein 78-kDa (GRP78) can restore ER function and reduce unfolded protein response (UPR) signaling. Therefore, we tested the hypothesis that expression of GRP78 could rescue G5G8 in db/db mice. Adenovirus encoding GRP78 was administered to db/db mice and the effect on hepatic G5G8 was determined. G5 and G8 proteins and biliary cholesterol were increased in the absence of changes in mRNAs encoding either protein.

However, TUDCA has also been shown to induce FGF15. In several models of bile acid feeding, FGF15/19 is stimulated in ileum and activates its receptor in liver to repress bile acid synthesis. Simultaneously, G5G8 and biliary cholesterol secretion are elevated. To determine if FGF15/19 had a direct effect on hepatic G5G8, we injected C57BL/6 mice with recombinant FGF19. CYP7A1 and CYP8B1 mRNA expression were both strongly suppressed, whereas G5G8 increased at both mRNA and protein levels.

In conclusion, G5G8 can be rescued in ob/ob and db/db mice through multiple mechanisms that include restoration of ER functions and FGF15/19 signaling. Counter regulation of CYP7A1, CYP8B1, and G5G8 by FGF15/19 allows for the maintenance of hepatic sterol elimination in the face of expanded bile acid pool.
Abstract 144: Zebrafish Larvae: A Model System for Early Stage Atherosclerosis?
Authors: Bandaru Manoj K , Ranefall Petter , Emmanouilidou Anastasia , Klingström Tiffany , Tao Lingjie , Wählby Carolina , Ingelsson Erik , den Hoed Marcel
Published: July 2021

Objectives:
Published results show that overfeeding zebrafish larvae on a high-cholesterol diet (HCD) can result in hypercholesterolemia and sub-endothelial lipid deposition in macrophages and other cell types. However, results are so far based on small samples, and the atherogenic response has been heterogeneous. We aim to use zebrafish larvae for large-scale, CRISPR-Cas9-based genetic screens, using results from genome wide association studies for coronary heart disease as a starting point. Firstly however, we need to ensure the model system is appropriate and robust. Therefore, we examined the effect of a high-energy diet (HED) and HCD on vascular lipid deposition in a larger number of larvae (n=241).

Methods:
Starting at 5 days post fertilization (dpf), ~30 larvae/tank were fed 2x/day on: 1) 5 mg control diet (CD; n=33); 2) 15 mg control diet (HED; n=90); or 3) 15 mg control diet enriched with 4% cholesterol (HCD; n=94). At 14-17 dpf, larvae were soaked in monodansylpentane cadaverase - a lipid staining dye - for 45 min, before imaging the dorsal aorta and caudal vein with a Leica SP5 confocal microscope. We used a custom written script in Cell Profiler to quantify the surface area of lipid deposits in the vasculature.

Results:
Manual annotation of vascular lipid deposition in 30 images (10 randomly selected images per dietary condition) allowed us to calculate the sensitivity (36%) and specificity (71%) of the Cell Profiler script. Subsequent analyses showed that HED (p=0.004) and HCD (p=0.001) fed larvae have significantly more vascular lipid deposition than CD fed larvae after adjusting for age, batch and vessel length. There was no difference in vascular lipid deposition between HED and HCD fed larvae (p=0.11).

Discussion and conclusion:
Our results confirm that zebrafish larvae represent a promising model system for early-stage atherosclerosis. In addition, they show that enriching the diet with cholesterol is not required to prompt atherogenesis.

Future directions:
In the next few months, we will examine if overfeeding also triggers vascular infiltration by macrophages, neutrophils and oxidized LDL cholesterol, and if atherogenesis can be prevented or reduced by treating larvae with statins and/or ezetimibe, using our new, automated imaging setup.
Abstract 176: Dietary PUFAs Enhance Macrophage Autophagy, Attenuate NLRP3 Inflammasome Activation, and Reduce Atherosclerosis
Authors: Shen Lulu , Shewale Swapnil V , Chung Chia-Chi , Boudyguina Elena , Parks John S , Zhu Xuewei
Published: July 2021

Progression of atherosclerosis leads to progressive macrophage autophagy deficiency, which enhances inflammasome activation and atherosclerosis. We previously showed that dietary enrichment with n-3 and n-6 polyunsaturated fatty acids (PUFAs) derived from botanical oils (Echium oil (EO) containing 18:4 n-3 and borage oil (BO) containing 18:3 n-6), reduced atherosclerosis similar to that observed with fish oil (FO) consumption, compared to the saturated/monounsaturated-enriched botanical palm oil (PO). We hypothesized that dietary PUFAs enhance autophagy, which in turn, dampens macrophage NLRP3 (Nucleotide-binding oligomerization domain leucine-rich repeat containing receptor protein 3) inflammasome activation, reducing macrophage inflammation and atherosclerosis. To test the hypothesis, we fed female low density lipoprotein receptor (LDLr) knockout mice diets containing 10% (calories) PO and 0.2% cholesterol, supplemented with an additional 10% of calories as PO, EO, BO or FO for 10-16 weeks before measurement of atherosclerosis, autophagy and inflammasome activation. Compared to their PO-fed counterparts, mice fed PUFAs enriched diets (EO, BO or FO) had increased LC3-II expression in peritoneal macrophage, aorta and liver, suggesting autophagic activation. Consistent with enhanced autophagy, plasma reactive oxygen species (ROS) were significantly lower in PUFA diet-fed mice, relative to PO-fed animals. Since ROS activates, but autophagy attenuates, NLRP3 inflammasome activation, we examined whether dietary PUFAs attenuate NLRP3 inflammasome activation in mice. We found that dietary PUFAs markedly inhibited inflammasome activation, shown by: 1) less IL-1β secretion from peritoneal macrophages after ATP, oxidized LDL, or palmitate-induced NLRP3 inflammasome activation, 2) less IL-1β secretion from liver explants in response to lipopolysaccharide (LPS), and 3) deceased caspase-1 cleavage in peritoneal macrophages and liver and attenuated caspase-1 activity in blood monocytes. In conclusion, our data suggest that dietary n-3 and n-6 PUFAs are equally effective in reducing atherosclerosis, in part, by activation of macrophage autophagy and attenuation of NLRP3 inflammasome activation.
Abstract 177: Ticagrelor, but not Clopidogrel, Protects the Heart Against Reperfusion Injury and Improves Remodeling After Myocardial Infarction
Authors: Ye Yumei , Perez-Polo Jose R , Nanhwan Manjyot K , Nylander Sven , Birnbaum Yochai
Published: July 2021

Background:
Clopidogrel (C) and Ticagrelor (T) are P2Y12 ADP receptor antagonists. In addition, ticagrelor inhibits adenosine cell uptake. In PLATO trial T reduced the incidence of the primary composite endpoint myocardial infarction, stroke or cardiovascular death over C in patients with acute coronary syndromes. Previous data show that 7d pretreatment with T limits infarct size (IS) in rats. We compared the effects of C and T, administered just before reperfusion on IS. We also assessed the effect of T and C, administered just before reperfusion and/or 6w oral treatment on cardiac remodeling. Methods: Rats underwent 30min coronary artery ligation. 1) At 25min of ischemia rats received intraperitoneal (IP) vehicle, T (10 or 30mg/kg), or C (12.5mg/kg). Area at risk (AR) was assessed by blue dye and IS by TTC staining 24h after reperfusion. 2) Rats received vehicle without (sham) or with (control) coronary ischemia, T (30mg/kg) IP (TIP), T (300mg/kg/d) oral for 6w, started a day after reperfusion (TPO), TIP+PO (TIPPO), or C (12.5mg/kg IP +62.5mg/kg/d PO for 6w). LV dimensions and function was assessed by echo at 6w. Results: 1) AR was comparable among groups. IS was 45.3±1.7% of the AR in the control group. T10 (31.5±1.8%; p=0.001) and T30 (21.4±2.6% p<0.001) significantly reduced IS, whereas C (42.4±2.6%) had no effect. Platelet aggregation in the controls was 64.7±1.3% and was comparable in T30 (24.9±1.8%) and C (23.2±1.8%) at 2h post reperfusion. T30 increased Akt, eNOS and ER1/2 phosphorylation 4h after reperfusion, whereas C had no effect. 2) Platelet aggregation at 1w oral treatment was 59.7±3.2% in the control group and was comparable in TIPPO (18.1±1.3%) and C (17.4±0.7%). Left ventricular ejection fraction was 77.6±0.9%*, 44.8±3.5%, 69.5±1.6%*, 69.2±1.0%*, 76.3±1.2%*, and 37.4±3.7% in the sham, vehicle, TIP, TPO, TIPPO and C treated group, respectively (*p<0.001 vs. vehicle). Left ventricular diameters at diastole and systole showed the same pattern. Conclusions: T, but not C, administered just before reperfusion protects against reperfusion injury. Oral T (in combination or not with acute treatment just before reperfusion) treatment for 6w improves heart function. C, despite achieving similar degree of platelet inhibition had no effect on remodeling.
Abstract 179: Ischemic Stroke in Atrial Fibrillation: 30-day Outcomes and Factors Associated with Severity
Authors: Ko Darae , Thigpen Jonathan , Henault Lori , Quinn Emily , Tripodis Yorghos , Berger Peter , Limdi Nita , Hylek Elaine
Published: July 2021

Background and Hypothesis:
Ischemic stroke (IS) in atrial fibrillation (AF) is associated with high mortality. Inflammation, endothelial dysfunction, and hypercoagulability, in addition to blood stasis in the left atrium, play a critical role in thrombogenesis in AF. Hyperglycemia and chronic kidney disease (CKD) are potent triggers for inflammation, oxidative stress, and thrombogenesis. Statins have been shown to possess anti-inflammatory, anti-oxidant, and anti-thrombotic properties. Accordingly, we assessed the hypothesis that statin use may modulate stroke severity in AF.

Methods:
Consecutive IS admissions were identified from 2006-2010. All events were subject to CT or MRI and assessed for functional independence at discharge using modified Rankin scale (mRS). AF was confirmed by ECG at presentation or within the prior 6 months in all cases. Covariates were abstracted from the medical record. To account for confounding by treatment, we used multivariable logistic regression analysis adjusted using inverse probability weighting.

Results:
We identified 1,030 AF-related IS; mean age was 77, 56% were female, mean CHA
2
DS
2
VASC score was 4.8 designating high baseline stroke risk. IS resulted in severe neurological deficit or death (mRS ≥ 4) for 69%; 21% died within 30-days. Severe stroke was associated with older age, diabetes, dementia, prior ischemic stroke, prior venous thromboembolism, and CKD (Table). Baseline statin use was associated with a 33% reduced risk of sustaining a severe stroke.

Conclusion:
Strokes in AF are associated with high morbidity and mortality. Clinical markers of thrombophilia, including prior IS, DVT, and PE, were significantly associated with severe strokes. Diabetes and CKD independently increased this risk. Statin use resulted in less severe outcomes. Advancing our basic understanding of these interrelated thrombogenic pathways will inform clinical interventions to reduce these devastating outcomes.
Abstract 184: Pulmonary Microvascular Thrombosis Enhances Extravasation via Myeloid Hypoxia-Inducible Factors
Authors: Evans Colin E , Branco-Price Cristina , Prodger Alice , Palazon Asis , Tyrakis Petros , Rundqvist Helene , Bendahl Par-Ola , Belting Mattias , Johnson Randall S
Published: July 2021

Introduction:
Mechanisms that regulate the positive association between thrombosis and metastasis are incompletely understood. It was hypothesised that thrombus formation stimulates a hypoxic response, which in turn promotes extravasation. The primary aim was to determine whether thrombosis of the pulmonary microvasculature (T
pm
) increases extravasation via myeloid (neutrophil and macrophage) hypoxia-inducible factor (HIF).

Methods and Results:
T
pm
was induced in wild type mice via tail vein administration of polystyrene microbeads (15μm diameter, 1000/mouse). T
pm
led to chronological increases in pulmonary HIF1α expression (P=0.01), HIF2α expression (P<0.01), neutrophil infiltration (P<0.05), and macrophage infiltration (P<0.05; 1-5days post-T
pm
vs. non-thrombosed vehicle controls, n=8/group/time point); these increases were comparable with changes observed following vena cava thrombosis (assessed via image analysis of immunostained tissue throughout). In wild type mice with circulating Lewis lung cancer cells (LLCs, 1million/mouse i/v), T
pm
led to increases in pulmonary fibrin deposition (P<0.0001), HIF1α expression (P<0.05), HIF2α expression (P<0.05), and LLC extravasation (P<0.0001; 14days post-LLCs vs. non-thrombosed controls, n=15/group). Using conditional HIFα knockout mice (vs. wild type littermates), it was shown that T
pm
-induced increases in pulmonary fibrin deposition and LLC extravasation were dependent upon HIF1α or HIF2α in neutrophils and macrophages; myeloid HIFs were also responsible for T
pm
-induced increases in pulmonary tumour proliferation and vascularisation (n=15/group). In human breast tumour microarrays (n=221), fibrin deposition was positively correlated with HIF2α expression (RS=0.22, P<0.001), while increases in HIF2α were associated with reductions in metastasis-free survival (HR=1.1, P<0.05).

Conclusions:
Thrombus formation in mouse pulmonary microvasculature enhances cancer cell extravasation via neutrophil- and macrophage-specific HIF1α or HIF2α. In human breast tumours, HIF2α is associated with increased fibrin deposition, and reduced survival. Pulmonary microvascular thrombosis can enhance cancer cell dissemination via myeloid cell-specific HIFs.
Abstract 199: Type 2 Diabetes Impairs Wound Healing By DNMT1-dependent Reduction of Hematopoietic Stem Cell Differentiation towards Monocytes/Macrophages and Skewing of the M1/M2 Polarization
Authors: Yan Jinglian , Tie Guodong , Khair Lyne , Filippova Elena , Messina Louis
Published: July 2021

Rationale:
People with Type 2 Diabetes Mellitus (T2DM) have a 25x higher risk of limb loss than non-diabetics due in large part to impaired wound healing. The mechanisms that cause impaired wound healing remain incompletely characterized.

Objective:
We hypothesize that T2DM impairs wound healing by epigenetic modifications in hematopoietic stem cells (HSC) that reduce their differentiation towards monocytes/macrophages and disrupts the balance in M1/M2 polarization during the three phases of wound healing.

Methods and Results:
Wounds were created on the back of mice. Wound healing was significantly slower in diabetic db/db than in WT mice. During the early inflammatory phase, db/db wounds exhibited a significant decrease in total macrophages and M1 macrophages. Then, total macrophages and M2 macrophages were decreased, while M1 macrophages increased in tissue formation phase. In the late tissue remodeling phase, total macrophages and M1 macrophages were persistently increased. The impaired wound healing phenotype of db/db mice was recapitulated in WT recipients which were resconstituted with db/db HSCs, demonstrating that the impaired differentiation of HSCs towards macrophages as well as their M1/M2 polarization was due to a cell autonomous mechanism. Epigenetic studies indicated that DNMT1-dependent hypermethylation of Notch1, Pu.1 and KLF4 in T2D HSCs was responsible for the impaired differentiation towards monocytes/macrophages as well as the skewed M1/M2 polarization. Knockdown of DNMT1 in HSCs from db/db mice transplanted into lethally irradiated WT mice led to improved wound healing by an increase in macrophage infiltration as well as a normalization of the M1/M2 polarization.

Conclusion:
This study indicates that the dynamic changes of macrophage concentration and M1/M2 polarization in wound healing are regulated at the level of HSCs. Moreover, T2DM impairs wound healing by inducing DNMT1-dependent reduction of HSCs’ differentiation towards macrophages and their M1/M2 polarization. This novel finding indicates that inflammation is regulated at the level of HSCs, which creates new opportunities to develop epigenetic modification related therapies for T2DM and potentially other conditions that result from dysinflammation.
Abstract 202: The Role of Neutrophil Extracellular Traps in the Pathogenesis of Pulmonary Hypertension.
Authors: Aldabbous Lulwah , Duluc Lucie , McKinnon Thomas , Howard Luke , Gibbs Simon , Wharton John , Morrell Nicholas , Toshner Mark , Southwood Mark , Pepke-Zaba Joanna , Abdul-Salam Vahitha , Wilkins Martin , Wojciak-Stothard Beata
Published: July 2021

Rationale:
Inflammation contributes to vasoconstriction and remodeling in pulmonary hypertension (PH), but the mechanisms are not fully understood. Recent data link neutrophil extracellular traps (NETs) and NET components with endothelial dysfunction, thrombosis, and lung injury. NETs, produced as a result of neutrophil cell death induced by inflammation or cytotoxic stress, comprise chromatin fibers associated with a number of proteins including myeloperoxidase and neutrophil elastase. We hypothesized that NETs contribute to pulmonary vascular remodeling in pulmonary hypertension. The aim of the present study was to measure NET markers in plasma of PH patients and to analyze their effects on human pulmonary artery endothelial cell (HPAEC) function in vitro.

Methods and Results:
Patients with prevalent chronic thromboembolic pulmonary hypertension (CTEPH) (n=26) and idiopathic pulmonary arterial hypertension (IPAH) (n=52) show increased plasma levels of NET components including myeloperoxidase, neutrophil elastase and circulating DNA, compared with plasma from sex- and age-matched healthy volunteers (n=24). Endarterectomy specimens from CTEPH patients show accumulation of NET-forming peptidylarginine deiminase 4+ (PAD4+) neutrophils. PAD4+ neutrophils are also abundant in and around plexiform lesions in IPAH lungs, compared to corresponding uninvolved regions of lung tissues from cancer patients. Incubation of NETs with HPAECs in vitro activates the transcription factor nuclear factor-kB and increases expression of pro-inflammatory cytokines and leukocyte-binding adhesion molecules. NETs also increase endothelial angiogenesis in vitro and induce plateletet aggregation, possibly due to increased expression and surface deposition of von Willebrand factor and P-selectin.

Conclusion:
CTEPH and IPAH patients show elevated NET markers in plasma and increased numbers of NET-forming neutrophils in the remodelled lung. NETs induce endothelial dysfunction in vitro and are likely to contribute to vascular remodelling in vivo. Inhibition of NET components or NET formation in vivo may represent a new therapeutic approach in the treatment of PH.
Abstract 203: Redox Regulation of MKP-1 and the Functional Reprogramming of Monocyte-Derived Macrophages by Metabolic Stress
Authors: Kim Hong Seok , Tavakoli Sina , Asmis Reto
Published: July 2021

Objective:
To determine the mechanisms through which MKP-1 deficiency in monocytes and macrophages promotes atherogenesis.

Methods and Results:
Transplantation of MKP-1-deficient (MKP
KO
) bone marrow into LDL-R
-/-
(MKP-1
LeuKO
) mice accelerated high fat diet (HFD)-induced atherosclerotic lesion formation. After 12 weeks of HFD feeding, MKP-1
LeuKO
mice showed increases in lesion size in both the aortic root and the aorta, despite reduced plasma cholesterol levels. Macrophage content was increased in lesions of MKP-1
LeuKO
mice compared to mice that received wt bone marrow. After only 6 weeks on a HFD, the
in vivo
chemotactic activity of monocytes was already significantly increased in MKP-1
LeuKO
mice. Macrophages isolated from MKP
KO
mice showed impaired autophagy, increased sensitivity to 7-ketocholesterol-induced apoptosis, and IL-4-induced M2 polarization of these macrophages was blocked. Importantly, macrophages in atherosclerotic lesion of MKP-1
LeuKO
mice showed increased macrophage apoptosis and decreased autophagy. Metabolic stress
in vitro
mimicked the functional phenotype of MKP-1 deficiency, and overexpression of MKP-1 protected macrophages from metabolic stress-induced dysfunction and reprogramming.

Conclusions:
Loss of MKP-1 activity promotes monocyte dysfucntion and the functional reprogramming of monocyte-derived macrophages. MKP-1 deficiency accelerates atherosclerotic lesion formation by hyper-sensitizing monocytes to chemokine-induced recruitment, predisposing macrophages to M1 polarization, impaired autophagy and oxysterol-induced cell death. Reduced MKP-1 levels and activity in blood monocytes may therefore be a sensitive biomarker of increased risk of atherosclerosis.
Abstract 22: Monocyte Chemotactic Protein-1 and Macrophage Colony-Stimulating Factor Are Strong Predictors of First-Time Myocardial Infarction in the General Population
Authors: Schiopu Alexandru , Bengtsson Eva , Goncalves Isabel , Nilsson Jan , Nordin Fredrikson Gunilla , Björkbacka Harry
Published: July 2021

Objective:
Monocytes/macrophages and neutrophils play important roles in the pathogenesis of cardiovascular disease. We hypothesized that mediators governing myeloid cell homeostasis might help predict the incidence of first-time acute myocardial infarction (AMI).

Methods:
We included 385 individuals who suffered an AMI during a median follow-up of 15.2 years, and 401 age and sex-matched controls that remained event free, recruited from the Malmö Diet and Cancer Study cohort. The participants had no documented coronary artery disease previous to inclusion. We performed parallel measurements of monocyte chemotactic protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF), CCL3, CCL4, CCL20, CXCL1, CXCL16, and CX3CL1 in plasma samples collected at baseline, by using a Proximity Ligation Assay.

Results:
MCP-1 presented a strong positive correlation (HR (95% CI): 2.07 (1.77-2.43), p<0.001), whereas M-CSF had a strong negative correlation with incident AMI (HR (95% CI): 0.49 (0.41-0.58), p<0.001). These relationships were independent of the classic Framingham risk factors (age, ex, smoking, total cholesterol, HDL, systolic blood pressure), diabetes, CRP, and of the other considered myeloid biomarkers. Addition of MCP-1 and M-CSF significantly improved the predictive ability of a model including the Framingham risk factors alone (c-statistic 0.82 [95% CI 0.78-0.85] with MCP-1 and M-CSF vs. 0.66 [95% CI 0.62-0.70], P<0.05). Addition of MCP-1 and M-CSF to the risk model led to a net reclassification improvement (NRI) of 0.51 (95% CI 0.42-0.60), and an integrated discrimination improvement (IDI) of 0.19 (95% CI 0.16-0.21). The model including MCP-1 and M-CSF was particularly effective in controls, leading to a 65% net re-classification into lower risk categories, and in subjects within the intermediate risk group (10-20%), where 70% of individuals were correctly up or down re-classified.

Conclusion:
MCP-1 and M-CSF have opposite relationships with the risk for AMI in the general population. Addition of MCP-1 and M-CSF significantly improves the predictive power of the Framingham risk model, leading to correct re-classification of a high percentage of individuals, particularly in the intermediate risk group.
Abstract 238: Distinct Vascular Effects of Celecoxib and Rofecoxib in vivo
Authors: Ricciotti Emanuela , Grosser Tilo , Grant Gregory R , Liu Shu-Lin , Lawson John A , Assoian Richard , FitzGerald Garret A
Published: July 2021

Randomized controlled trials of cyclooxygenase (COX)-2 selective NSAIDs revealed that these drugs confer a cardiovascular hazard, resulting mainly from suppression of COX-2 derived prostacyclin (PGI2).

Disruption of rodent COX-2-derived PGI2 in vascular cells and cardiomyocytes has recapitulated all elements of the cardiovascular hazard attributable to NSAIDs in humans.

To investigate further the impact of COX-2 inhibition in the vasculature, we examined the effects of therapeutic concentrations of two selective COX-2 inhibitors, celecoxib and rofecoxib, on thrombosis and vascular injury. Furthermore, we used RNA sequencing (RNA-Seq) to assess whether celecoxib and rofecoxib might elicit distinct genomic effects in mouse aorta.

Celecoxib (100 mg/Kg) and rofecoxib (50 mg/Kg) caused a selective and equipotent COX-2 inhibition as indicated by measuring thromboxane (TXB-M, an in vivo index of COX-1 activity) and prostacyclin (PGI-M, an in vivo index of COX-2 activity) urinary metabolites by mass spectrometry.

Both celecoxib and rofecoxib augmented platelet deposition and prolonged platelet disaggregation after laser-induced injury in the cremaster arterioles.

A similar effect was observed in mice lacking PGI2 receptor IP. There was no additional pro-thrombotic effect of the drugs on an IPKO background, consistent with suppression of COX-2 dependent PGI2 mediating the predisposition to thrombosis induced by celecoxib and rofecoxib.

Celecoxib, but not rofecoxib, caused a reduction of neointimal hyperplasia and the intima/media ratio of injured femoral artery at 3 weeks after wire injury.

RNA-seq analysis revealed 903 transcripts differentially expressed between celecoxib and control group and 3,127 transcripts between rofecoxib and control group (at a false discovery rate of 10%) in mouse aorta.

Thus, both celecoxib and rofecoxib cause a similar predisposition to thrombosis in mice.

Celecoxib, but not rofecoxib, reduces neointimal hyperplasia after vascular injury and COX-2 independent genetic actions might modulate this effect.

The predisposition to thrombosis and attenuation of the response to vascular injury by celecoxib recapitulates what has been found in humans.
Abstract 242: A Novel Transient Receptor Potential Ion Channel Vanilloid 4 Agonist Inhibits Monocytes Adhesion and Atherosclerosis via eNOS Activation
Authors: Xu Suowen , Liu Bin , Jin Felix Q , Yin Meimei , Koroleva Marina , Mastrangelo Michael , Jin Zheng Gen
Published: July 2021

Endothelial nitric oxide (NO) synthase (eNOS/NOS3)-derived NO represents an important vasculoprotective molecule in the cardiovascular system. The transient receptor potential ion channel vanilloid subtype 4 (TRPV4) mediates vascular mechanosensitivity, in part through an increase in blood flow-mediated NO production and endothelium-dependent relaxation. Here, we hypothesized that non-mechanical TRPV4 activation can mimic laminar flow-induced signaling and thereby limits monocyte adhesion and atherosclerosis. We observed that GSK1016790A, a novel, potent and specific agonist of TRPV4, mimics laminar flow, thereby inducing robust phosphorylation (Ser1177) of eNOS in a TRPV4 and calcium-dependent manner. Mechanistically, GSK1016790A induced eNOS phosphorylation, NO production and vasorelaxation via calcium/calmodulin-dependent kinase kinase (CaMKK)/AMP-activated protein kinase (AMPK) pathway. In functional experiments, GSK1016790A inhibited TNFα-induced monocytes adhesion to human endothelial cells by inhibiting the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), and this inhibition was abolished by an eNOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) or AMPK inhibitor Compound C. Mice given GSK1016790A showed increased eNOS (2.5- fold increase versus vehicle, n=12, P<0.001) and AMPK phosphorylation (2.1- fold increase versus vehicle, n=12, P<0.01) in the aorta. Finally, we demonstrated that oral administration of GSK1016790A inhibited atherosclerotic plaque formation in ApoE deficient mice fed a Western-type diet (13.27% ± 1.571%, versus vehicle 23.22% ± 1.130%, n=8, P<0.001). In conclusion, our data provide a novel mechanism whereby a TRPV4 agonist triggers NO production and suggests that TRPV4 agonists could be used as innovative vasculoprotective treatments of endothelial dysfunction-related diseases, such as atherosclerosis, hypertension and stroke.
Abstract 255: Loss of Alk5 in Smooth Muscle Cells (SMCs) Causes Aortic Aneurysms Through an Aberrant Tgfbr2 Signal
Authors: Yang Pu , Fu Chunhua , Hong Michael , Schmit Bradley , DeSart Kennyth , Oh Suk P , Berceli Scott A , Jiang Zhihua
Published: July 2021

TGF-β signaling disorder has emerged as a cause for aortic aneurysm formation. However, mechanistic details of this disorder remain poorly defined. This study sought to understand the TGF-β signaling propagation and its impact on aortic structural homeostasis under conditions of receptor deficiency. An inducible gene deletion system driven by Myh11-CreER was utilized for selective removal of receptors Alk5, Tgfbr2, or both in SMCs, termed Alk5iko, Tgfbr2iko, and Alk5iko Tgfbr2iko, respectively. Alk5-floxed (Alk5f/f) mice served as controls. Animals (male, 9-11 weeks of age) were followed up to six months following gene deletion. Alk5iko caused progressive aortic enlargement (ultrasound scan). In four weeks, 23% of animals (n=42) died from aortic rupture, with the survivals all showing severe aneurysmal degeneration at locations of ascending (100%), descending (43%) and/or suprarenal (50%) aortas. Surprisingly, Tgfbr2iko in age-matched males (n=26) was well tolerated. Neither rupture nor aortic dilation was noted in four weeks. Evidence of aortic pathology was limited to scattered elastic fiber breaks. To evaluate the role of TGF-β in aortic aneurysm formation in Alk5iko animals, we deleted Tgfbr2 simultaneously with Alk5. The additional Tgfbr2iko completely recused aortic rupture, attenuated aortic dilation from 68% to 25%, and significantly improved aortic histology. Compared to the Alk5f/f controls, Alk5iko, Tgfbr2iko, and Alk5iko Tgfbr2iko aortas all displayed a similar but reduced level of Smad2 phosphorylation at various time points, indicating that loss of the canonical TGF-β signal is not the primary driver for the aortic phenotype of Alk5iko aortas. Meanwhile, the Alk5iko aortas produced 3-fold more pERK than Alk5f/f controls, and this elevation was completely abolished by the additional Tgfbr2 deletion. Pharmaceutical treatment of the Alk5iko mice with ERK inhibitor RDEA-119 significantly inhibited aortic dilation and prevented aortic wall from degeneration while those treated with vehicle control exhibited aortic dilation and intimal/medial tears. In conclusion, loss of Alk5 in SMCs triggers a non-Alk5 but Tgfbr2 dependent signal that promotes aortic aneurysm formation through exacerbating ERK activity.
Abstract 256: Decreased Serum MiR-155 Expression is Associated with Increased Tumor Necrosis Factor-Alpha Levels in Patients with Abdominal Aortic Aneurysm
Authors: Afkhami Arash M , Nguyen Anthony T , Samadzadeh Kiana M , Rona Anjelica , Chun Kevin C , Lee Eugene S
Published: July 2021

Introduction:
The inflammatory response plays a crucial role in abdominal aortic aneurysm (AAA) pathogenesis and expansion. The cytokine tumor necrosis factor-alpha (TNF-α) and MicroRNA-155 (miR-155) are implicated in inflammatory diseases. Previous studies have found miR-155 regulates TNF-α expression in cardiovascular disease. However, the relationship between TNF-α expression and miR-155 in AAA disease is not fully understood. This study examines the relationship between serum TNF-α levels and miR-155 expression in AAA patients.

Methods:
MicroRNA’s were isolated from patient serum and quantified using Agilent BioAnalyzer 2100. MiR-155 levels were assessed using qPCR with Exiqon LNA primers and normalized using the 2-ΔΔCT method. Media was collected from the co-culture of control or AAA monocytes with endothelial cells. The supernatant was analyzed for the presence of TNF-α via Luminex
®
200
TM
analyzer. AAA is defined as ≥3.0 cm in maximum aortic diameter and measurement was obtained from abdominal ultrasound. Student’s t-test was used to compare AAA and control groups for statistical analysis.

Results:
Average aortic diameter for AAA group was 5.1±0.7 cm and 2.4±0.4 cm for controls. Q-PCR results showed miR-155 was significantly down-regulated in serum (p=0.03) in AAA subjects (n=5) versus non-AAA controls (n=4) (Figure 1A). Serum TNF-α levels from Luminex assays trended towards greater levels in AAA patients (n=4) vs. controls (n=3) (920.1±591.1 vs. 294.8±102.2 pg/mL, p=0.14) (Figure 1B).

Conclusion:
Patients with AAA may have more unregulated TNF-α activity due to decreased miR-155 expression compared to controls. Serum miR-155 may serve as a potential biomarker for patients at risk for aortic expansion leading to AAA disease.
Abstract 260: Identification of Micrornas Specifically Expressed in Isolated Cells of Human Abdominal Aortic Aneurysm
Authors: Pinet Florence , Spear Rafaelle , Hot David , Staels Bart , Chwastyniak Maggy , Amouyel Philippe , Haulon Stephan
Published: July 2021

Abdominal aortic aneurysm (AAA) is a vascular asymptomatic disease responsible for 4% of mortality in the elderly population. MicroRNAs (miRNA) have recently been shown to be potential biomarkers due to their stability in plasma.

The aim of this study was to investigate the potentiality of miRNAS as biomarkers of AAA by identifying miRNAs specifically expressed in the key cells present in the human AAA tissue.

The distribution of inflammatory cells such as smooth muscle cells (SMC), M1 and M2 macrophages, neutrophils, B and T lymphocytes and mast cells were located by immunohistochemistry in 20 human AAA biopsies, showing a specific distribution towards the aneurysmal aortic wall and the presence of adventitial tertiary lymphoid organs (ATLOs) in 10 samples. We isolated by laser microdissection (LMD) area enriched in aneurysmal SMC, M1 and M2 macrophages, and ATLOs and SMC from control aorta. RNA extracted from 2 samples of LMD-isolated cells was screened on human miRNAs microarray.

Out of the 850 human miRNAs, more than 200 miRNAs were detected in LMD-isolated aneurysmal cells, with 164 detected in ATLOs, 49 in SMC, and 87 miRNAs in macrophages. We selected the 3 miRs with the highest expression for ATLOs and 10 miRs (based on their miR-29b levels) for SMC and macrophages for validation by qRT-PCR in LMD-isolated samples (n=4).

We found that miR-15a-3p (0.1 fold) and miR-30a-5p (0.2 fold) were down-regulated and miR-489-3p up-regulated (2 fold) in ATLOs which was inversed for miR-489-3p in the whole aneurysmal aorta. Of the 10 miRNAs selected, six (miR-199a-3p and miR-451 upregulated and miR-24, miR-29a, miR-29b, and miR-29c downregulated) were modulated similarly in SMC and macrophages and whole aneurysmal aorta, except for miR-199-3p. Let-7f and miR-34a were similarly upregulated ed in both subtypes of macrophages and aneurysmal aorta.

Expression in the plasma of AAA (n=24) compared to PAD (n=18) patients was significantly down-regulated for miR-15a-3p (p = 0.03) and miR-30a-5p (p = 0.04) and upregulated for let-7f (p=0.048) and miR-29b (p=0.035).

In conclusion, this non-hypothesis driven screening of miRNAs expressed in isolated aneurysmal cells allowed to identify four miRNAs as potential AAA biomarkers.
Abstract 263: Evaluation of Survival Benefit of Statins in Patients with Pulmonary Hypertension and Chronic Obstructive Pulmonary Disease: A Propensity Score Matching Study
Authors: Holzhauser Luise , Hovnanian Ninel , Eshtehardi Parham , Mojadidi Khalid , Goodman-Meza David , Pamerla Mohan , Zolty Ronald
Published: July 2021

Background:
Pulmonary hypertension (PH) is a multifactorial disease characterized by endothelial dysfunction and inflammation. In line with this, pleiotropic effects of statins have been found beneficial in PH resulting in hemodynamic improvement. However, the role of statins in subgroups of PH, especially inflammation triggered chronic obstructive pulmonary disease (COPD) is unknown and their effect on mortality has not yet been studied.

Methods:
Consecutive patients ≥18 years with severe PH (pulmonary artery systolic pressure ≥60 mmHg) and preserved left ventricular function (ejection fraction ≥50%) defined by transthoracic echocardiograms were included from January 2002 to August 2012. Patients were divided into two groups based on being on statin therapy for 12 consecutive months after diagnosis of PH. Propensity score matching was performed for all baseline demographics, comorbidities, labs, lipid profile, and medications with ratio matching of 1 (treated) to 5 (untreated controls). Subgroup analysis was done based on COPD status. Study endpoint was 1-year all-cause mortality.

Results:
2,363 patients (age 71±16; 31% male) were included, 140 (6%) patients were on statin therapy. 1-year mortality for the entire population was 34%. Following propensity score matching, 137 patients on statin (statin group) and 625 patients not on statin (controls), all-cause mortality was significantly lower in statin group compared to controls (15.3% vs. 36.2%, HR 0.38 [95% CI 0.25, 0.60], p<0.001). After stratifying patients based on COPD status, while statins significantly reduced 1-year all-cause mortality in patients without COPD (HR 0.30 [95%CI 0.16, 0.56], p<0.001), patients with COPD did not show a survival benefit from statins (HR 0.54 [95%CI 0.28, 1.05], p=0.069).

Conclusions:
In this study, we identify statin therapy as an independent predictor of lower 1-year mortality in patients with severe PH but interestingly not in the subgroup of patients with COPD. This observation might be linked to the high severity of PH in our study population and less likely to the lack of anti-inflammatory effects. However the overall survival benefit in patients with severe PH is a novel and promising finding that needs to be confirmed in large randomized trials.
Abstract 272: Chronic Hyperlipidemia Alters the Population of Endothelial Progenitor Cells in Bone Marrow and Peripheral Circulation via Both ROS-dependent and Independent Mechanisms
Authors: Liu Dylan Z , Cui Yuqi , Liu Jason Z , Liu Lingjuan , Li Xin , Xiao Yuan , Zhang Jia , Xie Xiaoyun , Hao Hong , He Guanglong , Parthasarathy Sampath , Zhu Hua
Published: July 2021

Background/Aims:
Bone marrow (BM)-derived endothelial progenitor cells (EPCs) make significant contribution to the function and integrity of vasculature. The number of EPCs is significantly decreased in hyperlipidemic patients. Reactive oxygen species (ROS) and oxidative stress were considered an important mechanism for the development of atherosclerosis in hyperlipidemia. The present study was to determine the role of ROS production in the changes of EPC population in chronic hyperlipidemia.

Methods and Results:
EPC numbers and ROS formation in BM and blood were determined in wild-type (WT) male C57BL/6 mice and hyperlipidemic LDL receptor knockout (LDLR-/-) mice with high fat diet for 4 months. Intracellular blood, extracellular BM and blood ROS production was significantly increased in hyperlipidemic LDLR-/- mice that was effectively blocked with N-acetylcysteine treatment. Hyperlipidemia produced complex changes in EPC populations in BM and blood. The c-Kit+/CD31+ cell number was significantly decreased in BM and blood, and the numbers of CD34+/CD133+ cells and Sca-1+/Flk-1+ cells were significantly decreased in blood without change in BM, which were not affected by inhibition of ROS production. Interestingly, blood CD34+/Flk-1+ cell number was significantly increased in hyperlipidemic mice that was prevented when ROS formation was inhibited.

Conclusions:
Chronic hyperlipidemia produced significant and complex changes in EPC populations in both BM and circulation through both ROS-dependent and ROS-independent mechanisms in mice.