bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 604: Influence of Perivascular Adipose Tissue on Vasodilation in Metabolic Syndrome
Authors: Kagota Satomi , Maruyama Kana , Iwata Saki , Wakuda Hirokazu , Yoshikawa Noriko , Nakamura Kazuki , Shinozuka Kazumasa
Published: March 2022

Metabolic syndrome facilitates the development of cardiovascular disease due to atherosclerosis. We propose that chronic oxidative-nitrative stress is linked to the development of vascular dysfunction in response to nitric oxide (NO) in the coronary and mesenteric arteries of SHRSP.Z-
Lepr
fa
/IzmDmcr (SHRSP.ZF) rats, an animal model of metabolic syndrome. Perivascular adipose tissue (PVAT), which is located outside blood vessels, has now been recognized as playing a role in vascular function. We assessed the role of PVAT in vascular dysfunction observed in SHRSP.ZF rats with metabolic syndrome.

We used SHRSP.ZF rats at 13, 18, and 23 weeks, and observed obesity, hyperlipidemia, glucose intolerance, and hypertension from 13 weeks until 23 weeks of age. Relaxation induced by acetylcholine and sodium nitroprusside were unchanged in isolated mesenteric arteries of SHRSP.ZF rats at 13 weeks of age, but were impaired at 18 and 23 weeks of age compared to those in control Wistar-Kyoto rats (WKY). The relaxation induced by both agonists was greater in the mesenteric arteries enveloped by PVAT than in those without PVAT in SHRSP.ZF rats at 18 and 23 weeks of age. The relaxation in the arteries with PVAT in SHRSP.ZF rats were at similar levels as those in the arteries without PVAT in age-matched WKY. In contrast, no difference was observed in the relaxation levels in the arteries with and without PVAT in SHRSP.ZF rats at 13 weeks of age and in WKY at all ages. Histological analysis of PVAT showed that adipocytes in PVAT of SHRSP.ZF rats at 20 weeks of age were larger than those observed in WKY. Impaired NO-dependent vasodilation was also observed in aortas of SHRSP.ZF rats at 23 weeks of age compared to that in age-matched WKY, but the relaxation was not altered by the presence of PVAT.

SHRSP.ZF rats with metabolic syndrome showed impairment in the NO-dependent pathway in resistant arteries, with increasing age/exposure to metabolic abnormalities. PVAT may help in the regulation of vasodilation to compensate for the impaired vasodilation observed under pathophysiological conditions, such as metabolic syndrome.
Abstract 605: Association of Apolipoprotein B with Impaired Insulin Sensitivity and Secretion Is Mediated by Reduced Fat Clearance in Obese Subjects
Authors: Lamantia Valerie , Bissonnette Simon , Saint-Pierre Nathalie , Gauthier Dany , Wassef Hanny , Faraj May
Published: March 2022

OBJECTIVE:
Human data from our lab and others have demonstrated that elevated concentrations of apoB-lipoproteins, measured as plasma apoB, are associated with insulin resistance (IR), hyperinsulinemia and the development of type 2 diabetes. However, the underlying mechanism to date remains unknown. We recently reported that LDL, the major form of apoB-lipoproteins, directly impairs the hydrolysis and storage of triglyceride-rich lipoproteins
ex vivo
in human white adipose tissue. Therefore, we hypothesized that the association between apoB and IR and compensatory increase in insulin secretion (IS) is mediated by delayed postprandial TG clearance
in vivo
in obese subjects.

METHODS:
We performed a Botnia clamp on normoglycemic obese men (N=14) and postmenopausal women (N=16) (BMI ≥ 27 kg/m2, age ≥ 45 years). First phase, 2
nd
phase and total IS were measured during a 1-hour intravenous-glucose tolerance test (IVGTT) while insulin sensitivity was measured during a 3-hours hyperinsulinemic euglycemic clamp that followed. On a separate date, postprandial plasma clearance of TG was measured over 6 hours following the ingestion of a
13
C-triolein-labeled high-fat meal.

RESULTS:
Plasma apoB correlated positively with IR (r=0.15, p=0.037), 2
nd
phase IS (r=0.15, p=0.039) and delayed TG clearance (AUC
6hrs
of total plasma TG: r=0.64, p<0.001; AUC
6hrs
of
13
C-plasma TG: r=0.77, p<0.001). Moreover, AUC
6hr
of total plasma TG was associated with higher IR (r=0.16, p=0.042), 1
st
phase IS (r=0.20, p=0.023), 2
nd
phase IS (r=0.16, p=0.044) and total IS (r=0.16, p=0.046). Similarly, a positive association existed between delayed
13
C-labeled plasma TG clearance (AUC
6hrs
) and IR (r=0.41, p=0.046). A gender difference existed as TG clearance was mainly associated with IR in women (r=0.41, p=0.046) whereas it was mainly associated with 2
nd
phase IS in men (r=0.41, p=0.046). As hypothesized, correcting for TG clearance eliminated the association of plasma apoB with IR and IS.

CONCLUSION:
The association of apoB with insulin resistance and secretion may be secondary to LDL-mediated delayed TG clearance by adipose tissue in humans.
Abstract 611: Blocking CD40-TRAF6 Signaling is a Novel Therapeutic Target in Obesity-Associated Insulin Resistance
Authors: Seijkens Tom , Chatzigeorgiou Antonios , Zarzycka Barbara , Engel David , Poggi Marjorie , van den Berg Susan M , van den Berg Sjoerd A , Soehnlein Oliver , Winkels Holger , Beckers Linda , Lievens Dirk , Driessen Ann , Kusters Pascal , Biessen Erik , Garcia Martin Ruben , Klotzsche-von Ameln Anne , Gijbels Marion J , Noelle Randolph J , Boon Louis , Hackeng Tilman M , Martin Schulte Klaus , Xu Aimin , Vriend Gert , Nabuurs Sander B , Chung Kyoung-Jin , Willems van Dijk Ko , Rensen Patrick C , Gerdes Norbert , de Winther Menno P , Block Norman L , Schally Andrew W , Weber Christian , Bornstein Stefan R , Nicolaes Gerry A , Chavakis Triantafyllos , Lutgens Esther
Published: March 2022

The immune system plays an instrumental role in obesity and insulin resistance. Here we unravel the role of the co-stimulatory molecule, CD40, and its signaling intermediates, TNF-Receptor-Associated-Factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40-/- mice in DIO displayed worsened insulin resistance, as compared to wild type mice. This was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8+ T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40-/- mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance, and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this improved insulin sensitivity, reduced AT inflammation and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity, whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our newly developed compound may provide a novel therapeutic option in obesity-associated insulin resistance.
Abstract 619: A Role for Macrophage Lipopolysaccharide Binding Protein in Atherosclerosis Development
Authors: Sallam Tamer , Gilliland Thomas , Ito Ayaka , Rong Xin , Kim Jason , van Stijn Caroline , Chamberlain Brian T , Jung Michael E , Chao Lily C , Jones Marius , Wu XiaoHui , Su Grace L , Tangirala Rajendra K , Tontonoz Peter , Hong Cynthia
Published: March 2022

Introduction:
The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. Knockout of LXRs in macrophages greatly accelerates the development of atherosclerosis whereas pharmacological LXR activation has potent athero-protective effects. Previous studies have shown that deletion of LXR target genes in hematopoietic cells can have opposing effects on atherosclerosis development. For example, deletion of ABCA1 using bone marrow transfer in atherogenic mouse models showed increases atherosclerotic plaque burden where as knockout studies of ABCG1 and Aim1 decreased lesion formation in bone marrow transplantation experiments owing to an increase in macrophage apoptosis.

Rationale:
We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis.

Methods & Results:
We demonstrate that the lipopolysaccharide binding protein (LBP), a secreted glycoprotein, is an LXR target gene in macrophages. Treatment of mouse peritoneal macrophages with modified LDL or oxysterols induces LBP expression, an effect that was lost in LXRαβ-/- macrophages, consistent with a role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies using WT or LBP-/- donors and LDLR-/- recipients. After 18 weeks of western diet feeding atherosclerotic lesion burden was assessed by en face and aortic root section analysis. LDLR-/- mice receiving LBP-/- bone marrow had markedly smaller lesions compared to those receiving WT (P<0.0001 in en face analysis; P<.01 in root section analysis). Furthermore, loss of bone marrow LBP expression led to a strong increase in apoptosis in atherosclerotic lesions as determined by TUNEL staining. In vitro studies with isolated macrophages and stable cell lines showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading.

Conclusions:
The LBP gene is a macrophage LXR target that promotes foam cell survival and atherogenesis. Our study identifies LBP as a potential diagnostic and therapeutic target in human cardiovascular disease.
Abstract 620: Identification of Novel ABCA1 Mutations in Families With Tangier Disease
Authors: Brunham Liam , Kang Martin , Van Karnebeek Clara , Rabkin Simon , Stockler Sylvia , Cassiman David , Hayden Michael
Published: March 2022

Tangier disease is an extremely rare, autosomal recessive disorder caused by mutations in the ABCA1 gene, and is characterized by near absence of plasma high density lipoprotein cholesterol, cholesterol accumulation in multiple tissues, peripheral neuropathy and accelerated atherosclerosis. Here we report three kindreds with Tangier disease. We describe two novel coding mutations in the ABCA1 gene that result in premature truncation of the protein as well as a novel intronic variant that leads to creation of a cryptic splice site acceptor resulting in aberrant mRNA splicing and premature truncation of the protein. We also show that this mutation arose de novo in the proband, the first demonstration of a pathogenic de novo mutation in ABCA1 associated with Tangier disease. Our findings expand the list of pathogenic mutations in ABCA1 and our understanding of how mutations in this gene lead to abnormal cellular and physiological phenotypes.
Abstract 63: MerTK and Mfge8-Dependent Efferocytosis Orchestrates Postischemic Cardiac Healing Through VEGF-A and Il-10 Regulation
Authors: HoWangYin Kiave-Yune , Yin Min , Cochain Clément , Gueguen Marie , Vilar José , Loyer Xavier , Mallat Ziad , Boulanger Chantal M , Silvestre Jean-Sébastien
Published: March 2022

Inflammation orchestrates tissue remodeling after myocardial infarction (MI). Efferocytosis is mainly carried out by the membrane protein tyrosin kinase mertk (MerTK) and the milk fat globule epidermal growth factor (Mfge8) and may balance inflammation resolution after MI. We reasoned that efficient MerTK and Mfge8-dependent clearance of apoptotic debris by infiltrated myeloid cells is critical to the angiogenic and regenerative fine-tuning of the ischemic cardiac tissue. We first demonstrated that the left ventricle ejection fraction assessed by echocardiography and the number of capillaries were decreased by around 1.5-fold in MerTK -/-, Mfge8 -/- and double deficient mice when compared to wild-type animals (WT), 14 days after the onset of ischemia (p<0.01, n=10 per group). These effects were associated with an increased infarct size, fibrotic area as well as apoptotic cells number and were also observed in irradiated WT mice transplanted with bone marrow-derived cells isolated from MerTK -/-, Mfge8 -/- and double deficient animals. In cardiac tissue of WT CD45.1 recipient mice, the number of infiltrated CD45.2 MerTK -/-/Mfge8 -/- bone marrow-derived cells was similar to that of CD45.2 WT cells. In addition, the amount of Ly6Chigh and Ly6Clow monocytes as well as that of macrophages was unaffected in infarcted heart of MerTK -/-/Mfge8 -/- bone-marrow transplanted compared to WT mice. In contrast, deficient efferocytosis was related to an increased number of cardiac annexinV-positive microparticles and a downregulation of the pro-angiogenic growth factor VEGF-A and the anti-inflammatory cytokine IL-10 protein levels. Notably, FACS-sorted Ly6Clow monocytes from ischemic cardiac tissue of MerTK -/-/Mfge8 -/- mice expressed lower levels of VEGF-A when compared to WT cells. Hence, inhibition of efferocytosis leads to an adverse cardiac remodeling after MI through phenotypic alterations of phagocytic cells.
Abstract 64: Pancreatic ß-Cell--Specific Deletion of ABCA1 and ABCG1 Perturbs Glucose Metabolism and Increases Adiposity in Mice
Authors: Cochran Blake J , Hou Liming , Moore Benjamin M , Sultana Afroza , Tall Alan R , Barter Philip J , Rye Kerry-Anne
Published: March 2022

Background:
Evidence suggests that pancreatic lipid accumulation causes β-cell dysfunction. Mice with β-cell specific ABCA1 deletion and global knockout of ABCG1 have increased β-cell sterol levels, impaired glucose tolerance, raised fasting plasma glucose levels and impaired glucose-stimulated insulin secretion. However, as mice with global deletion of ABCG1 also have low adipose tissue mass and do not become glucose intolerant or insulin resistant when challenged with a high fat diet, that study did not provide an insight into the specific impact of β-cell dysfunction on glucose metabolism.

Aim:
Investigate the effects of isolated β-cell-specific deletion of ABCA1 and ABCG1 on glucose metabolism in mice.

Methods:
β-cell specific knockout mice were generated by crossing Ins2-cre and Abca1fl/flAbcg1fl/fl mice. Animals were maintained on a chow diet from the time of weaning until 16 weeks of age. Body weight and food intake were monitored weekly. At 16 weeks of age, mice were subjected to glucose (2 g/kg) and insulin (1 U/kg) tolerance tests. Blood glucose and insulin levels were measured with a glucometer and by RIA, respectively. Plasma MCP-1 and IL-6 levels were measured by ELISA. Fat and muscle mass were assessed by MRI.

Results:
Islet ABCA1 and ABCG1 expression was reduced by 76% and 70%, respectively, in knockout mice compared to control. ABCA1 and ABCG1 expression was comparable in all other tissues. Control and knockout animals had comparable islet mass and insulin content, but the knockout mice were markedly glucose intolerant (AUC 2649±230 vs 1539±189) and had reduced fasting insulin levels (0.67±0.18 vs 1.11±0.17 ng/mL). Insulin levels increased 1.5 fold in response to a glucose challenge in control animals, while no increase was observed in knockout animals. Both knockout and control animals were insulin sensitive. Despite similar weight and food intake, the knockout mice had a 28% increase in adiposity and a 10% reduction in muscle mass and plasma IL-6 and MCP-1 levels were increased 3.5- and 5-fold, respectively, relative to the control animals.

Conclusion:
β-cell specific deletion of ABCA1 and ABCG1 in mice reduces both acute and chronic insulin secretion, impairs glucose metabolism, alters body composition and increases systemic inflammation.
Abstract 643: Absence of Four-and-a-Half LIM Domain Protein 2 Decreases Atherosclerosis in ApoE-/- Mice: Role of Monocytic Immune Cells
Authors: Ebrahimian Talin , Simon David , Simeone Stefania , Lemarie Catherine A , Heidari Maryam , Mann Koren K , Wassmann Sven , Lehoux Stephanie
Published: March 2022

Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial and vascular smooth muscle cells. It negatively regulates endothelial cell survival and migration, but its role in atherogenesis is unknown. To investigate the role of FHL2 in atherosclerosis, FHL2-deficient (FHL2-/-) mice were crossed with ApoE-deficient (ApoE -/-) mice, to generate ApoE/FHL2-/- mice. After 7 weeks of high fat diet, ApoE/FHL2-/- mice had significantly smaller (P<0.05) atherosclerotic plaques than ApoE-/- mice in the aortic sinus (0.14±0.02 vs. 0.29±0.04 mm2), the brachiocephalic artery (0.03±0.008 vs. 0.07±0.01 mm2) and the aorta (6.9±0.9 vs 10.3±1%), assessed by oil red O staining. This was associated with enhanced collagen (16±2 vs 8.6±3 %) and smooth muscle cell (4.5±0.8 vs 1.8±0.5%) contents within the plaques of ApoE/FHL-2-/- mice. Moreover, macrophage content within plaques was reduced 2-fold in ApoE/FHL2-/- vs ApoE-/- mice (P<0.05). This could be explained, in part, by the 40% reduction in aortic ICAM-1 mRNA and 55% reduction in VCAM-1 protein expression in the plaque. Furthermore, aortic gene expression of Cx3cl1 and Ccl5 was increased in ApoE/FHL2-/- vs ApoE-/- mice, suggesting that different monocyte populations might be recruited to the plaques. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of pro-inflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- vs ApoE-/- mice (8±3 vs 18±4%). Furthermore, mRNA levels of Cx3cr1 were 2-fold higher in monocytes from ApoE/FHL2-/- mice compared with ApoE-/- mice. Finally, we also investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. ApoE-/- or ApoE/FHL2-/- mice were lethally irradiated and transplanted with ApoE-/- or ApoE/FHL2-/- bone marrow. After recovery and 7 weeks of high fat diet, both chimeric groups developed smaller plaques than ApoE-/- mice transplanted with ApoE-/- bone marrow. These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting pro-inflammatory chemokine production, adhesion molecule expression, and pro-inflammatory monocyte recruitment.
Abstract 163: Induction of Lysosomal Biogenesis in Macrophages Reduces Atherosclerosis in an Autophagy-dependent Manner
Authors: Sergin Ismail , Bhattacharya Somashubhra , Emanuel Roy , Razani Babak
Published: March 2022

Recent reports of the proatherogenic phenotype of mice with a macrophage-specific autophagy deficiency have renewed interest in the role of the autophagy-lysosomal system in atherosclerosis. Lysosomes have the unique role of processing both exogenous material such as excess atherogenic lipids and endogenous cargo that includes dysfunctional proteins and organelles via autophagy. Previously we demonstrated that oxidized LDL and cholesterol crystals, two of the commonly encountered lipid species in the atherosclerotic plaque, create a profound lysosomal and autophagy dysfunction in cultured macrophages. Overexpression of TFEB, a transcription factor that is the only known master regulator of lysosomal and autophagy biogenesis, in macrophages initiates a robust prodegradative response including induction of lysosomal and autophagy genes. This in turn ameliorates several deleterious effects of the lipid-mediated dysfunction, namely the blunting of inflammasome activation, enhancing cholesterol efflux, and accelerating the degradation of protein aggregates. Our in vitro data suggest that the induction of a lysosomal biogenesis program in macrophages can have atheroprotective effects. Indeed, myeloid-specific TFEB overexpression in mice significantly reduces atherosclerotic plaque burden as well as plaque complexity as gauged by reduced necrotic core and markers of apoptosis. Interestingly, this protection is autophagy-dependent since these TFEB-overexpressing mice on a background of myeloid-specific autophagy (ATG5)-deficiency no longer demonstrate plaque reduction. Mechanistically, this indicates that suppression of the inflammasome and enhancement of cholesterol efflux and protein aggregate removal is dependent on the TFEB-autophagy axis. Taken together, our data support the notion that harnessing the prodegradative response in macrophages via TFEB can be atheroprotective and provides the impetus to evaluate mechanisms by which macrophage lysosomal and autophagy biogenesis can be modulated therapeutically.
Abstract 167: High Dietary Fructose Does not Exacerbate the Detrimental Consequences of a HF Diet on Basilar Artery Function
Authors: Tumer Nihal , Toklu Hale Z , Muller-Delp Judy , Sakarya Yasemin , Oktay Sehkar , Kirichenko Nataliya , Matheny Michael , Carter Christy , Morgan Drake , Strehler Kevin , Scarpace Philip J
Published: March 2022

There is a strong correlation between the consumption of sugar- or fructose-enriched products and obesity. Moreover, high dietary fructose without added fat and in the absence of obesity induces leptin resistance and exacerbates subsequent HF-induced obesity. Elevated leptin is associated with arterial stiffness. Thus, we hypothesized that the consequences of a high-fat/high-fructose (HF/F) diet would be more detrimental than a high-fat (HF) or chow diet on functional and structural changes in the basilar arteries. Male Sprague Dawley rats were fed either with chow, HF (30%) or HF/F (30%/44%) diets for 12 weeks (N=8-10/group). The basilar artery was cannulated in a pressurized system (90cm H2O) and vascular contractile responses to KCl (30-120 mM) and endothelin (10-11-10-7 M), and relaxation to acetylcholine (ACh) (10-10-10-4 M), and diethylamine (DEA)-NONO-ate (10-10-10-4 M) evaluated and artery wall thickness and eNOS immunofluroescence determined. At death, leptin, blood lipids, and asymetricaldiamethyl arginine level (ADMA) were assayed in serum. Basilar artery contractility was unchanged, but both HF and HF/F diets resulted in decreased relaxation responses compared with chow to endothelium dependent (Ach logEC50: -6.48, chow; -5.65, HF; 5.79HF/F, p<0.05) and endothelium independent relaxation (DEA: -5.94;-4.45;-4.49, p<.05). Increased basilar artery wall thickness was observed only in HF group (22±1.2 vs 27±1.6μm, p<.05). Both diets increased serum ADMA levels (.36±.03 HF; .42±.05 HF/F vs .26±.02 chow, p<.05), 50% decreased eNOS immunofluorescence (arbitrary units, p<.05), and increased adipose tissue (10.7±.5 HF; 12.7±.8 HF/F vs 7.8±.3 chow, p<.05). HF/F increased triglycerides (157±32 mg/dl) and leptin (10.4±1.8ng/ml) greater than with HF (65±15; 6.6±.6) or chow diet (63±14; 4.1±.3). In conclusion, both HF diets impaired basilar artery relaxation, potentially due to decreased eNOS synthesis and increased ADMA. Although, leptin and triglycerides were greater with HF/F diet only the HF increased wall thickness. These data suggest that the presence of high fructose in a HF diet does not exacerbate the detrimental consequences of a HF diet on basilar artery function.
Abstract 197: Statin Therapy Is Associated With Higher
MTHFR
Methylation Levels in a Stroke Cohort
Authors: MacLeod Mary , Pogoryelova Oksana , Horgan Graham , Haggarty Paul
Published: March 2022

Introduction:
Epigenetics refers to changes in gene function that occur by gene methylation, histone modification or RNA mechanisms. Many of these are heritable, but can also be altered by lifestyle and medication. Genome-wide hypomethylation is associated with atherosclerosis and hyperhomocysteinemia, and specific genes can be hyper or hypomethylated in vascular disease. Statins have pleiotropic effects on the vasculature, and may affect DNA methylation.

Hypothesis:
The methylation levels of genes implicated in vascular disease are altered by treatment with common cardiovascular medications.

Methods:
189 individuals from a well characterised stroke cohort and controls were included. 44 were established on statin therapy, 49 on aspirin and 28 on an ACE inhibitor. The following genes implicated in vascular disease were investigated:
LINE1, SOD3, ALOX15, ERa, MTHFR
,
PITX2, IFNGR1, TNFa, IL10,
and
INS
. Methylation status was assessed by bisulphite conversion of DNA followed by PCR and pyrosequencing. Each methylation site was analysed individually and mean methylation level across all sites within a gene calculated. Statistical analysis for the effect of drug therapy was based on logistic regression and ANOVA.

Results:
One significant association was found: subjects on statin therapy had higher
MTHFR
methylation levels than those not receiving statin (p=0.002). Statistical analysis adjusted for stroke effect (p=0.025) remained significant. Correction for multiple testing was not performed as the candidate genes are linked in pathways. There was no effect of aspirin or ACE inhibitor on methylation status of any of the genes tested.

Discussion:
MTHFR
plays an important role in homocysteine and folate metabolism, providing methyl groups for DNA methylation. High levels of homocysteine are a risk factor for cardiovascular disease and there is published evidence that statin therapy results in lower plasma homocysteine levels. This finding requires further investigation in a larger cohort with a defined dose and duration of treatment. If this finding is replicated, there may be value in exploring the mechanisms linking statins,
MTHFR
methylation and homocysteine levels and the implications for primary and secondary prevention of vascular events.
Abstract 207: Reduced Vascular Density in Human Adipose Tissue in Diabetes is Associated with Reduced Angiogenesis and De-differentiation of Endothelial Cells
Authors: Bronson Haynes A , Hatcher Margaret A , Wohlgemuth Steven D , Lieb David C , Dobrian Anca D
Published: March 2022

Emerging evidence suggests that impaired adipose tissue (AT) angiogenesis leads to metabolic dysfunction. The de-differentiation of endothelial cells (EC) into a mesenchymal phenotype (EndoMT) in AT vasculature could be a potential contributor to dysfunctional EC including impaired angiogenesis. One of the major drivers of EndoMT is TGF, and both macrophages and T cells infiltrating AT in obesity could contribute to EndoMT via TGF production. The objective of this project was to characterize angiogenesis and the EC phenotype in human AT vasculature. We examined functional angiogenesis and gene expression in omental (OM) AT from obese T2D subjects (BMI>33, HbA1c>6.5%) and non-diabetic (ND) subjects (BMI>33). Sprouting angiogenesis of AT explants embedded in matrigel was 1.5-fold higher in ND vs. T2D (p<0.05). Also, capillary and arteriolar density assessed as CD31 and α-SMC immuno-positivity, was 2-fold higher in ND vs. T2D. Using a Human Angiogenesis PCR Array we found that in T2D vs. ND OM AT, there was >2-fold down-regulation in expression of genes involved in cell proliferation and matrix remodeling (JAG, ID1, COL4A3, TIMP1 and CTGF) (p<0.05). In conclusion, in T2D in obesity there is a reduction in vascular density that can be explained in part by the reduced angiogenic potential. Next, we tested the hypothesis that a potential contributor to reduced angiogenesis is de-differentiation of EC in AT vasculature. We determined the molecular phenotype of EC isolated from human AT. Indeed, EC (CD34
+
31
+
) isolated from ND and T2D obese AT had significantly >10-fold up-regulation of mesenchymal genes (TGF-β, fibroblast specific protein-1 (FSP1), and Twist1) compared to dermal human microvascular endothelial cells (HMVEC) (p<0.001). Also, EC isolated from AT displayed a prominent oxidative metabolism compared to dermal HMVEC that utilized primarily glycolysis. This phenotype was similar to the one of TGF treated dermal HMVEC. Since glycolysis is required for angiogenic ECs, the low glycolytic flux of AT EC could contribute to impaired angiogenesis. In conclusion, EC isolated from obese and T2D patients show increased expression of mesenchymal markers and a metabolic shift towards the oxidative metabolism, which may explain reduced angiogenesis.
Abstract 214: Balancing Innate Immunity Activation and Death Signals for Vascular Regeneration
Authors: Ospino Frank , Chanda Palas , Cooke John , Wu Donna , Kaiser William , Mocarski Edward , Sayed Nazish
Published: March 2022

Background:
Our recent discoveries have shown that innate signaling supports effective nuclear reprogramming (Lee & Sayed et al. Cell) and transdifferentiation to ECs (Sayed et al. Circ). By activating innate immunity (via TLR3), viral vectors used for reprogramming cause global epigenetic changes that favor an environment conducive for reprogramming. Indeed, activation of innate transcription factors (TFs) enhances EC regeneration, however, activation of the same pathway could induce: 1) apoptosis via activation of Casp8; and 2) programmed necrosis by receptor-interacting protein kinase 3 (RIP3), when Casp8 is absent.

Hypothesis:
We speculate that death pathways that are activated by these TFs compromise reprogramming. We previously showed that disruption of Casp8 led to mid-gestational death of mice due to unleashing of RIP3-dependent death pathways, preventing the formation of vascular endothelium and hematopoietic cells. By contrast, when Casp8
-/-
mice were crossed with Rip3
-/-
, the DKO mice developed normally with no stem cell defect. We hypothesize that these pathways exist as biological constraints on epigenetic plasticity and that pharmacological or genetic ablation could enhance EC reprogramming.

Results:
To test our hypothesis, we transdifferentiated WT and Casp8
-/-
Rip3
-/-
DKO MEFs using our protocol, employing an innate immunity modifier and EC growth factors. Intriguingly, our preliminary data showed that DKO MEFs transdifferentiated to induced-EC (iECs) with >10-fold higher yield when compared to WT (yields ~30%). Genetic and functional assays showed that iECs generated from DKO MEFs were comparable to WT, indicating that removal of apoptotic pathways did not lead to aberrant reprogramming. Moreover, pharmacological inhibitors of death receptors when combined with our small molecule cocktail showed a similar increase in iEC generation in human fibroblasts.

Conclusion:
This study is a first step toward development of a regenerative strategy for PAD on the use of ECs derived from small molecules and growth factors without use of viral vectors encoding TFs. We intend to derive an effective and feasible technology for therapeutic transdifferentiation for ischemic syndromes to promote healing with tissue rather than a scar.
Abstract 277: Perivascular Adipose Adiponectin Correlates with Symptom Status of Patients Undergoing Carotid Endarterectomy
Authors: Sharma Gaurav , Tao Ming , Ding Kui , Yu David , King William , Deyneko Galina , Wang Xiasong , Longchamp Alban , Schoen Frederick J , Ozaki C. Keith , Semel Marcus E
Published: March 2022

Introduction:
Recent symptoms stand as a major determinant of stroke risk in carotid stenosis patients, likely reflective of atherosclerotic plaque destabilization.

Hypothesis:
In view of emerging links between vascular and adipose biology, we hypothesized that human perivascular adipose characteristics associate with carotid disease symptom status.

Methods:
Clinical history, carotid plaques, blood, and subcutaneous and perivascular adipose tissues were prospectively collected from consented patients undergoing carotid endarterectomy (CEA). Nine adipose associated biologic mediators (adiponectin, IL-1 beta, IL-6, IL-8, leptin, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, resistin, and tumor necrosis factor) protein levels were quantified, and compared in patients with symptomatic (n=15) versus asymptomatic (n=19) disease. Bonferroni correction was performed for multiple testing (alpha/9=0.006).

Results:
Symptomatic patients had 1.9-fold higher perivascular adiponectin levels (median [interquartile range] of symptomatic versus asymptomatic patients was 478.4 ng/ml [315.4-603.9 ng/ml] versus 251.9 ng/ml [212.7-365.4ng/ml], p=0.005). Other circulating, subcutaneous, and perivascular biomarkers, as well as microscopic plaque characteristics, did not differ between symptomatic and asymptomatic patients.

Conclusions:
Symptomatic and asymptomatic CEA patients display a tissue-specific difference in perivascular adipose adiponectin. This biomarker signature, which was not seen in plasma or subcutaneous compartments, supports a potential local paracrine relationship to vascular disease processes which may relate to stroke mechanisms. Future study of these adipose based paracrine effects may point to novel therapeutic targets and inform patient selection for preventative interventions.
Abstract 308: Corticosteroid Therapy Benefits Septic Mice With Adrenal Insufficiency but Harms Septic Mice Without Adrenal Insufficiency
Authors: Ai Junting , Guo Ling , Zheng Zhong , Wang Shuxia , Li Xiang-an
Published: March 2022

Rationale:
Corticosteroid (CS) therapy is frequently used in septic patients. However, the efficacy of CS therapy is highly controversial. The heterogeneity of septic patients, the technical difficulties in identifying adrenal insufficiency and a lack of adrenal insufficiency animal model present significant barriers to address this longstanding clinical issue.

Objectives:
In this study, we utilized scavenger receptor BI (SR-BI) null mice as the first relative adrenal insufficiency animal model to evaluate the efficacy of CS therapy and assess the pathogenesis caused by adrenal insufficiency.

Methods:
Cecal ligation and puncture (CLP) was conducted on SR-BI-/- and wild type control mice, and then CS was administered 2 or 18h post CLP.

Measurements and Main Results:
We demonstrated that CS supplementation protects against CLP-induced sepsis in SR-BI-/- mice but causes more septic death in wild type controls. Importantly, we identified a cocktail of CS that provides more efficient protection than an individual CS, and the septic mice could be rescued 18h post CLP by the CS cocktail; we further demonstrated that adrenal insufficiency impairs both innate and adaptive immunity.

Conclusions:
This preclinical study provides a proof-of-concept that corticosteroid benefits septic patients with adrenal insufficiency but harms septic patients without adrenal insufficiency, and suggests that it is necessary to determine the status of adrenal insufficiency and only prescribe corticosteroid for septic patients with adrenal insufficiency.
Abstract 31: Functional Impact of Inflammatory Cell-Originating HDL-miR-223 Communication in vivo
Authors: Wiese Carrie B , Roteta Leslie A , Zhu Wanying , Landstreet Stuart R , Vickers Kasey C
Published: March 2022

Atherosclerosis is associated with endothelial dysfunction and inflammation, and high-density lipoproteins (HDL) play critical roles in both. HDL transport a wide-variety of proteins, nucleic acids, and small molecules, which likely confer many of HDL’s alternative functions, including its anti-inflammatory and anti-atherogenic properties. Using in vitro studies, we demonstrated that macrophages export miR-223 to HDL and subsequently, HDL delivers functional miR-223 to human coronary artery endothelial cells (HCAEC). Moreover, intracellular adhesion molecule 1 (ICAM-1), a miR-223 target, was found to be down-regulated by HDL-miR-223 at a specific target site within ICAM-1’s 3’ untranslated region. These in vitro findings support the potential of a HDL-miRNA communication pathway that may contribute to atherogenesis. To validate HDL-miRNA communication in vivo, bone-marrow transplant (BMT) studies were completed with Mir223-/- and C57B/6J (WT) mice. To demonstrate that functional HDL-miR-223 is delivered to recipient cells/tissues in vivo, BM from WT mice was transplanted into irradiated Mir223-/- mice. Strikingly, we found that HDL-miR-223 communication was restored after BMT, as evidenced by significantly increased miR-223 levels on HDL and in the liver, white adipose, aortic endothelium, and skeletal muscle. Conversely, HDL-miR-223 intercellular communication was depleted by transplanting BM from Mir223-/- into WT mice, and resulted in significant loss of miR-223 on HDL and in many tissues. In both BMT studies, miR-223 changes led to altered expression of many target genes in multiple tissues. Most importantly, these studies identified a novel miR-223 target gene which we experimentally validated, vesicle transport through interaction with t-SNAREs homolog 1A (VTI1A), which was decreased and increased in the aortic endothelium from each BMT study, respectively. VTI1A has been previously characterized as a SNARE protein that contributes to cytokine secretion and membrane lipid dynamics. As such, HDL likely mediates an inflammatory cell-originating miR-223 communication pathway that suppresses inflammation and cytokine secretion in the aortic endothelium, thus antagonizing atherosclerosis.
Abstract 33: Role of Epsins in Regulating LPS-Induced Sepsis
Authors: Pasula Satish , Brophy Megan L , Tessneer Kandice L , Hahn Scott , McManus John , Zhu Hua , Chang Baojun , Dong Yunzhou , Cai Xiaofeng , Song Hoogeun , Wu Hao , Chen Hong
Published: March 2022

Background:
Sepsis is caused by a deleterious host response to infection, which is primarily responsible for further injury of host tissue and cause of organ dysfunction. However, the underlying regulatory mechanisms are still not fully understood. Our goal is to define the novel role of epsins in regulating sepsis.

Methods and Results:
We engineered global (iDKO) and endothelial cell-specific (EC-iDKO) epsin deficient mice. When treated with lethal dose of LPS, epsin deficient mice were completely protected from LPS-induced septic death. These mice also exhibited decreased expression of tissue damage biomarkers and recruitment of neutrophils and macrophages to lungs compared to wild type (WT) suggesting that epsin deficiency mitigates sepsis induced tissue injury. Epsin deficiency further reduced expression of proinflammatory cytokines and adhesion molecules in the lungs suggesting that loss of epsin attenuates LPS-induced inflammatory responses. TAT complex production was also decreased in iDKO mice compared to WT indicating diminished coagulation and thrombin production. Knocking down of epsins in HUVECs resulted in reduced cell surface Tissue Factor (TF) expression. Loss of epsin in mice protected against loss of Thrombomodulin (TM), which is downregulated by sepsis. Mechanistically, loss of epsin inhibited LPS-induced TM internalization, while LPS treatment induced the ubiquitination of TM. Furthermore, co-IP of full length epsin 1 or epsin 1 without the UIM domain and TM demonstrated that UIM is required for the interaction between epsin 1 and TM. Collectively, we show that epsin-deficiency upregulates TM surface protein expression by preventing its internalization and subsequent degradation and inhibits heightened TF expression and activation under chronic inflammatory conditions such as that induced by LPS exposure.

Conclusions:
Our findings demonstrate that epsins play a key role in regulating coagulation and provide fundamental information on the modulation of the ratio of TM/TF in various thrombotic diseases including sepsis. Furthermore, we demonstrate loss of epsin protects mice against LPS-induced sepsis, suggesting a crucial role for epsins in promoting the development of LPS-induced sepsis.
Abstract 350: The Pro-atherosclerotic Milieu Converts a Sub-population of Murine Peripheral T Regulatory Cells to a Non-suppressive Th1-like Foxp3+IFNγ+ Phenotype.
Authors: Butcher Matthew J , McGary Christopher M , Filipowicz Adam R , Galkina Elena V
Published: March 2022

CD4+ T cell subsets play diverse roles in inflammation but may also display limited plasticity. The existence of a population of Foxp3+ T regulatory (Treg) cells that co-expresses Interferon-γ (IFNγ) has been demonstrated in several models of inflammation. In atherosclerosis, circulating Treg content inversely corresponds with atherogenesis, suggesting that inflammation might affect peripheral Treg (pTregs) maintenance. Thus, we sought to examine the fate of pTregs within atherosclerotic Apolipoprotein E-deficient (Apoe-/-) mice. Foxp3+IFNγ+ T cells were elevated within the aorta, spleen, and peripheral lymph nodes (PLNs) of atherosclerotic Apoe-/- versus C57BL/6 mice. Further phenotyping of Foxp3+IFNγ+ T cells revealed variable expression of Treg and Th1 markers, including low CTLA4, GITR, CD25 expression, equivalent PD1, CD73, FR4, expression and elevated Tbet, CD119, CXCR3, and CCR5 expression. To determine whether Foxp3+IFNγ+ T cells arise from de novo differentiation or peripheral conversion, we performed fate tracking experiments with co-adoptively transferred naïve T cells and pTregs, or lineage tracing Foxp3yfp-cre+ R26RtdTomato+ pTregs, to aged Apoe-/- mice. Naïve T cells failed to generate Foxp3+IFNγ+ T cells. In contrast, adoptively transferred Tregs maintained Foxp3 expression (50-60%) or adopted a Foxp3+IFNγ+ T phenotype (40-50%) within the spleen, peri-aortic lymph nodes, and aorta. To test the functionality of IFNγ+ Tregs, we isolated IFNγ+ pTregs, IFNγ- pTregs, and Foxp3GFP+ Tregs for T cell suppression assays. While IFNγ- pTregs and Foxp3GFP+ pTregs were equivalently suppressive, IFNγ+ pTregs failed to adequately suppress T cell proliferation. Together these results demonstrate that atherogenic inflammation affects the maintenance of pTregs, resulting in the generation of a subset of dysfunctional Foxp3+IFNγ+ T cells.
Abstract 363: Aortic Walls From Cadavers With Atherosclerosis Show Autoimmune Reactions: Exudation of Proteoglycans, Globulins And Interleukin-17, and Infiltration of Plasma Cells.
Authors: Nagareda Tomofumi , Hirata Nobuaki , Ohsawa Masahiko , Tanaka Sayaka , Kosakai Yoshio , Kimata Koji , Hatano Sonoko
Published: March 2022

Athersclerosis is known to be caused by inflammation of aortic intima. In order to elucidate the mechanism of atherosclerosis, we investigated the inflammated changes of media of the ascending aorta associated with coronary artery disease in 44 biopsy specimens. The findings made us confident that inflammation occurs not only in the intima but also in the media of the atherosclerotic aorta, and also made us suspect that atherosclerosis may be caused by an autoimmune reaction. Here, we investigate proteoglycans, globulins, lymphoid cells, and interleukin-17 (IL-17) infiltrating into the wall of atherosclerotic aorta from cadavers, to confirm this idea.

Twenty cadavers from 32 to 93 years old were studied. We divided the subjects into 4 groups with 5 cases each: 1-20% as a slight group, 20-50% as a mild group, 50-75% as a moderate group, and 75% or more as a severe group, in terms of the percentage of atheromatous lesion from the ascending to the abdominal aorta. We immunohistochemically stained the ascending aortic wall with antibodies: LY111 for chondroitin-4-sulfate, 2B1 for Versican core protein, LCA and CD4 for lymphocytes, IL17 for cells positive for this, CD68 for macrophage, and CD38 for plasma cells. In addition, using the aortic wall, we delegated the protein electrophoresis demarcation and the measure of the values of IL-17 to a laboratory corporation.

Chondroitin-4-sulfate was stained diffusely positive in the entire wall. Versican showed characteristic patchy figure in the media. The numbers of patchy figures were significantly more in the mild and moderate groups than those in the slight and severe groups. In a similar manner, the numbers of plasma cells in the intima and media were significantly more in mild and moderate groups than those in the slight and severe groups. Protein electrophoresis showed the state of chronic inflammation. Among the 4 groups, the values (pg/mg) of IL-17 of mild (307.572±91.341) and moderate (183.027±30.926) groups were significantly increased compared with those of slight (108.310±43.008) and severe (69.812±17.465) groups.

The changes of the infiltration of plasma cell, the exudation of proteoglycans and also the increase of IL-17 in the wall make it more confident that atherosclerosis is related to an autoimmune reaction.
Abstract 364: Pioglitazone Inhibition of Vascular Smooth Muscle Cell Proliferation is Associated with Activation of AMP-activated Protein Kinase but not Peroxisome Proliferator-Activated Receptor Gamma
Authors: Osman Islam , Pichavaram Prahalathan , Pyla Rajkumar , Segar Lakshman
Published: March 2022

Atherosclerosis and restenosis after angioplasty are associated with exaggerated proliferation of vascular smooth muscle cells (VSMCs). Pioglitazone (PIO), an antidiabetic drug that belongs to the family of peroxisome proliferator-activated receptor gamma (PPARg) agonists, reduces intimal hyperplasia after coronary angioplasty in diabetic and nondiabetic subjects. However, the molecular mechanisms by which PIO regulates VSMC phenotype remain unclear. The objective of the current study is to determine the role of AMP-activated protein kinase (AMPK, a cellular energy sensor) versus PPARg in PIO-induced changes in VSMC phenotype. Exposure of human aortic VSMCs to PIO followed by immunoblot analysis revealed a robust increase in the phosphorylation of AMPK (Thr172) and its downstream effector, Acetyl-CoA carboxylase (p < 0.05, n = 3), as a function of concentration (3 to 30 μM) and time (1 to 48 hr). Notably, PIO (30 μM, 48 hr) activation of AMPK remained essentially the same after PPARg downregulation (~90%) by target-specific siRNA (500 pmoles), suggesting lack of dependency on PPARg. In addition, PIO activation of AMPK was accompanied by induction of mitochondrial membrane depolarization with a trend toward an increase in AMP/ATP ratio (~40%), as revealed by tetramethylrhodamine methyl ester (TMRM) fluorescent redistribution & LC-MS/MS MRM analysis, respectively. Importantly, PIO pretreatment (30 μM, 30 min) resulted in significant inhibition of PDGF (30 ng/ml, 48 hr)-induced VSMC proliferation by ~85% (cell count) and the associated increases in proliferative markers (cyclin D1 and pRb) by > 95% (p < 0.05, n = 3). Downregulation of AMPKa1 (~80%) by target-specific AMPKa1 siRNA (500 pmoles) revealed that PIO-mediated AMPK activation inhibited mTORC1 signaling by > 95% & upregulated cell cycle inhibitor p27Kip1 by ~40%, thus accounting for PIO-mediated changes in VSMC phenotype. In vivo, PIO administration (10 mg/Kg/d, oral gavage) for 3 weeks in CJ57BL6 mice mitigated neointimal growth by ~60% in guide wire-injured femoral artery (p < 0.05, n = 8). In conclusion, the present study provides the first direct evidence for VSMC-specific AMPK activation by PIO, which may in part contribute to its vasoprotective effects in nondiabetic state.
Abstract 374: Does Aortic Stiffness Precede, Follow or Occur Simultaneously with Aortic Atherosclerosis?
Authors: Roldan Paola , Greene Ernest , Roldan Pablo , Rodriguez Rodrigo , Roldan Carlos
Published: March 2022

Background:
Aortic stiffness (AoS) and atherosclerosis (AoA) are common in systemic lupus erythematosus (SLE). Whether AoS precedes, follows, or occurs simultaneously with AoA is unknown.

Methods:
75 SLE patients (68 women, age 37±12 years) and 26 age-and-sex matched healthy controls underwent transesophageal echocardiography for assessment of AoS and AoA of the proximal, mid and distal thoracic aorta. Brachial blood pressures were obtained and matched in time with measurement of aortic diameters. AoS was assessed using the Pressure-Strain Elastic Modulus (PSEM) = [k(sBP–dBP)/(SD-dD/dD)] where k = 133.3 is the conversion factor from mmHg to Nm
-2
[Pascal units (U)], sBP=systolic blood pressure, dBP=diastolic blood pressure, SD=systolic diameter and dD=diastolic diameter. AoS was defined as the mean PSEM of controls +1.5SD = >9.4U. AoA was assessed using two-dimensional and M-mode images and defined as intima-media thickening (mean intima-media thickness of controls +2SD = >0.96mm) or plaques (>50% focal wall thickening as compared with surrounding walls).

Results:
Patients had higher PSEM and intima media-thickness than controls (8.14±4.25 U and 0.83±0.29 mm vs 5.97±2.31 U and 0.66±0.15 mm, respectively, both p≤0.002 ). Patients had higher frequency of abnormal AoS (29%) and AoA (35%) than controls (8% for both AoS and AoA) (both p≤0.03). In SLE patients and in relation to age, AoS and AoA occurred at similar rates (p=0.42) (
Figure 1).

Conclusion:
AoS occurs simultaneously with AoA in SLE patients.