bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 344: Incretin-Loaded Nanocarriers for Glucose Control, Therapy and Imaging of Atherosclerosis
Authors: Maiseyeu Andrei , Barajas Alma , Hesse Derek , Venkataraman Arjun , Rajagopalan Sanjay
Published: March 2022

Background:
Glucagon-like peptide-1 (GLP-1) is a key regulator of glucose control via interaction with the GLP-1 receptor (GLP-1R). GLP-1R is G-protein coupled receptor that signals through PKA-cAMP widely expressed on various cells including innate immune cells.

Objective:
To develop MRI-visible nanoparticles (NPs) carrying a GLP-1 mimetic Liraglutide (NP-Lira) and to test the hypothesis if NP-Lira accumulates in atherosclerosis and exerts anti-inflammatory and lipoprotein effects.

Methods and Results:
High-fat fed ApoE
-/-
mice were injected with a single dose of Lira-NP or Naked-NP followed by an oral glucose challenge. Glucose control was significantly improved with NP-Lira but not with NP-Naked (
Fig. A
). Fast protein liquid chromatography demonstrated that VLDL-apoB levels were significantly decreased by 66% at 2 hrs with NP-Lira vs. NP-controls together with reduction of triglycerides (
Fig. B, C
). Imaging of aortic plaque on a 9.4 T magnet revealed that NP-Lira accumulated in atherosclerosis as evidenced by increase in plaque CNR by 256% as compared to pre-injection (
Fig. D
).
In vitro
experiments demonstrated potent effects of NP-Lira vs NP-Naked in reducing pro-inflammatory gene expression and potentiation of
in vitro
cholesterol efflux
via
c-AMP dependent mechanisms.

Conclusion:
Nanocarriers containing GLP-1 reduce post-prandial lipids and glucose control. We postulate that their concomitant accumulation within plaque may result in favorable effects on inflammation and cholesterol efflux.
Abstract 347: Chronic Kidney Disease Modulates Vascular Smooth Muscle Cell Phenotype and Increases de Novo Venous Intimal Hyperplasia
Authors: Monroy M Alexandra , Fang Jinhua , Li Shan , Ferrer Lucas , Lee Iris , Birkenbach Mark P , Choi Eric T
Published: March 2022

Background:
Vascular access dysfunction associated with arteriovenous grafts and fistulas contributes significantly to the morbidity and mortality of end-stage renal disease (ESRD) patients receiving hemodialysis. The mechanism involved in the development of peri-anastomotic intimal hyperplasia, a major cause of the arteriovenous access dysfunction, is poorly elucidated. We hypothesized that the uremic condition associated with ESRD promotes a pathophysiological vascular smooth muscle cell (VSMC) phenotype that contributes to accelerated development of intimal hyperplasia in blood vessels, both de novo and following the arteriovenous anastomosis.

Methods and Results:
We examined the effect of culturing human VSMC with human uremic serum on its phenotype. We utilized quantitative real-time PCR to measure the expression of the VSMC-specific contractile marker genes, and found that these genes were reduced 50-80% in VSMC exposed to serum from hemodialysis patients as compared to serum from patients with normal renal function. Chromatin immunoprecipitation assays demonstrated that there were also significant changes in epigenetic markers associated with contractile gene promoters in VSMC cultured in uremic conditions. Moreover, there was a 60% increase in proliferation rate in cells exposed to uremic conditions, with no change in the levels of apoptosis as compared to cells exposed to non-uremic conditions. Interestingly, uremic serum from ESRD patients inhibited PDGF-induced migration of VSMC. We also examined the histopathology of veins from patients with normal kidney function and chronic kidney disease (CKD). Indeed, we found significant de novo venous intimal hyperplasia exists in CKD and ESRD patients prior to any surgical manipulation as compared to veins from patients with normal kidney function.

Conclusion:
CKD alters VSMC phenotype in vitro and contributes to intimal hyperplasia formation in vivo resulting in the pathogenesis of vascular access dysfunction in ESRD patients.
Abstract 350: Metabolism of Nitrite and Sulfide in the Aging Cardiovascular System
Authors: Rajpal Saurabh , Kolluru Gopi K , Pardue Sibile , Shen Xinggui , Kevil Christopher G
Published: March 2022

Introduction:
Hydrogen Sulfide (H2S) and Nitric Oxide (NO) are gaseous signaling molecules that affect many pathophysiological functions like vasodilation, anti-inflammation, cytoprotection, anti-apoptosis, mitochondrial respiration and angiogenesis. These actions suggest that the relationship between these gasotransmitters could be involved in the cardiovascular aging response.

Objective:
We examined the age dependent regulation of H2S and NO metabolites in mice.

Methods:
Levels of H2S and NO metabolites were measured in various organs of C57BL/6J wild type mice at age 6, 12 and 24 weeks. The monobromobimane (MBB)-HPLC method was used to measure various sulfide pools. NO metabolites were measured using NO chemiluminescence. One-way ANOVA with bonferroni post-testing was used to compare metabolites between age groups. A p value of ≤ 0.05 was considered significant.

Results:
In the heart, lung and kidneys there was a significant increase in total and free sulfide levels with age over 24 weeks. In the heart, bound sulfide and acid labile sulfide remained stable with age. In the kidney, the bound sulfide increased with age and acid labile sulfide remained stable. In the lung, bound sulfide remained stable and acid labile sulfide remained stable with age. In the aorta and mesenteric artery, tissue total sulfide levels decreased with age. Lastly, the ratio between total H2S versus NO significantly increased with age in the heart and kidney.

Conclusion:
Our study reveals novel relationships between cardiovascular H2S and NO metabolism during aging. The increased H2S/NO ratio during aging may indicate important biochemical changes that affect cardiovascular pathogenesis.
Abstract 352: Locally Deployed Perivalvular Leptin Promotes Thickening of Aortic Valve Leaflets Associated with Remodeling of the Left Ventricle in ApoE-Deficient Mice
Authors: Ben Zvi Danny , Fisch Sudeshna , Ozaki Keith C , Schoen Fredrick J , Schneiderman Jacob
Published: March 2022

Background:
Locally available leptin is involved in atherogenesis and remodeling of the arterial wall. Recently, we demonstrated its contribution to aortic aneurysm formation in mice, and hypothesized that leptin may also drive the process of aortic valve stenosis (AVS) by promoting medial degeneration in the valve cusps.

Methods:
ApoE-deficient mice underwent left mini-thoracotomy to allow the deployment of a miniature polyglycolic-base film designed for sustained release of mouse leptin (20μg), or placebo film on the aortic surface at the peri-valvular region. All mice received a high fat diet, and underwent serial cardiac echo examinations thereby, recording the diameter of the ascending aorta, blood flow through the aortic valve, and left ventricle morphology, throughout 45 days’ follow up.

Results:
Longitudinal echocardiography assessment in leptin vs placebo treated mice revealed progressive dilatation of the proximal ascending aorta, corroborating postmortem histological evidence for local fragmentation of elastic lamellas, and depletion of smooth muscle cells in the aortic wall. Leptin receivers manifested increased aortic valve leaflets’ thickening (p=0.05). This was associated with local proliferation α-SMA and TGF-β positive cells, suggestive of valve interstitial cells (VICs). Leaflets’ thickening correlated with in vivo echocardiography data indicating increased peak systolic velocity across the aortic valve (p=0.02). Also, increased left ventricle wall thickening (p=0.05), and decreased ventricular cross section area (p=0.01) were evident in leptin receiving mice.

Conclusions:
These preliminary results indicate that peri-aortic, peri-valvular leptin application, simulating local leptin synthesis, promotes remodeling of the aortic valve in ApoE-deficient mice, by unknown mechanisms. Leaflet thickening is associated with remodeling of the left ventricle, thus simulating and potentially providing a model for AVS pathogenesis and secondary effects.
Abstract 358: Genetic Determinants of Lower-Extremity Claudication in South Asians
Authors: Damrauer Scott M , Zhao Wei , Fairman Ronald M , Rader Daniel J , Saleheen Danish ,   PROMIS Investigators
Published: March 2022

OBJECTIVES:
The prevalence of peripheral arterial disease (PAD) in South Asian populations is less than that reported in populations of Western descent despite higher rates of MI and diabetes. This suggests that there may be a different underlying genetic predisposition to PAD in South Asian individuals. This study was designed to investigate the genetic determinants of PAD in a large Pakistani cohort.

METHODS:
We examined the association between single nucleotide polymorphisms (SNPs) and clinical symptoms of claudication in participants of the Pakistan Risk of Myocardial Infarction Study (PROMIS), a multicenter case-control study of MI in Pakistan, using its 1000-Genome imputed data set.

RESULTS:
724 (4.8%) of the 15,570 individuals enrolled in PROMIS had symptoms of claudication. These subjects had an average (SD) age of 56 (9.7) years and 536 (74%) were male. To assess the role of known MI risk loci, 250 kBP regions flanking the 45 reported MI risk SNPs were examined for their association with claudication. No SNP in any of the known MI risk loci met the pre-determined Bonferroni p-value threshold for significance (<1x10-5). Likewise, a composite risk score based on reported MI risk related SNPs was not associated with claudication symptoms despite a highly significant correlation with MI (p=4.7x10-34). In genome wide analysis no SNP achieved significance with a p-value <5 x 10-8. There were, however, three SNPs with p-values <5 x 10-7, suggestive of potential associations with claudication: rs7580101 (p=1.1 x 10-7), rs4655022 (p=1.4 x 10-7), and rs57859654 (p=2.1 x 10-7). Interestingly, rs7580101 occurs within an intron of the gene for CD59, a complement inhibitor that has been demonstrated to modulate atherosclerosis in multiple mouse models. The CD59 gene has not been previously reported as a MI or PAD risk locus.

CONCLUSIONS:
These findings argue for a different genetic risk profile for PAD and CAD in South Asians, supporting the differences in PAD prevalence between this population and those of Western descent. Additionally, they identify numerous novel potential risk loci for PAD. Replication studies are currently underway to validate these findings.
Abstract 377: Angiotensin II Drives Murine Ly-6chi Monocyte Trafficking from the Spleen to the Aneuvrismal Aorta Through B Cells
Authors: Mellak Safa , Loyer Xavier , Esposito Bruno , Ait Oufella Hafid , Tedgui Alain , Mallat Ziad , Potteaux Stephane
Published: March 2022

Abdominal aortic aneurysm (AAA) is widespread among elderly people and results in progressive expansion and rupture of the aorta with high mortality. AAA is histologically characterized by medial degeneration and various degrees of chronic adventitial inflammation. In particular, macrophages initially accumulate within the site of aneurysm. Early depletion of circulating monocytes protects against the disease. Two monocyte subsets with different migratory behaviours and functions have been described in mice, ‘inflammatory’ Ly6C+ monocytes (classical) and ‘patrolling’ Ly6C- monocytes (nonclassical). In this study, we aim to understand and clarify the role of monocyte-derived cells in AAA development.

We monitored monocyte mobilisation up to 28 days in a mouse model of inflammatory AAA, which consists of implanting subcutaneous osmotic pumps delivering Angiotensin II (AngII) at a rate of 1000 ng/kg/min into C57BL6 ApoE-/- male mice between 8 and 12 weeks old. We found that AAA initiation is translated by a rapid and temporary mobilisation of classical monocytes at day 3-6 after AngII followed by non classical monocytes at days 7-9. We showed that classical monocytes are mobilized from the spleen, then recruited in the abdominal adventitia of ApoE-/- in response to AngII treatment as monitored through a pulse labelling method, along with an increased proteasic activity at the abdominal vessel wall as seen by fluorescence tomography. Importantly, we found that classical monocyte number positively correlated to stages of AAA. Splenectomy performed prior to AAA initiation prevented classical monocyte mobilization at day 3-6 after AngII and significantly protected against AAA. We found similar results in ApoE-/- Rag2-/- mice reconstituted with B cell-depleted splenocytes and treated with AngII, suggesting that splenic B cells are responsible for early deleterious mobilization of classical monocytes.

Taken together, the data show that splenic classical monocytes directly participate to AAA pathology and could be exploited to improve the prognosis of patients harbouring AAA.
Abstract 383: Paired Immunoglobin-Like Receptor B Regulates Platelet Activation
Authors: Fan Xuemei , Shi Panlai , Dai Jing , Lu Yeling , Wang Kemin , Zhang Junfeng , Zheng Junke , Liu Junling
Published: March 2022

Introduction:
Many of the immunoglobulin (Ig) super family proteins are expressed on platelet surface, and involve in the regulation of platelet activation. Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptors LILRB2 or LILRB3, bearing extracellular Ig-like domains and immunoreceptor tyrosine-based inhibitory motifs, is broadly expressed and plays important roles in immune responses, neuron axonal regeneration and hematopoietic processes. The expressions and functions of PIRB in platelet remain unknown.

Hypothesis:
PIRB regulates platelet activation.

Methods:
PIRB intracellular domain deletion mice (PIRB-TM) were used to elucidate the functions of PIRB in platelet activation.

Results:
We found that LILRB2 and LILRB3 (or PIRB) expressed in human (or mouse) platelets. The Increasing megakaryocytes ratio in bone marrow and mild thrombocythemia were observed in PIRB-TM mice. Deficiency of PIRB facilitated agonists-induced platelet aggregation and spreading on immobilized fibrinogen (Fg). The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also enhanced. Further signaling analysis revealed that PIRB bound to SHP1 and SHP2. And collagen related peptide (CRP)-induced tyrosine phosphorylation of LAT, SLP76 and PLCγ2 increased in PIRB-TM platelet. The phosphorylation of FAK Y397 and integrin β3 Y785, but not integrin β3 Y773, was also enhanced in PIRB-TM platelet spread on immobilized Fg. We also found that one of the PIRB ligands angiopoietin like protein 2 (Angptl2) was highly expressed and stored in platelet α-granule, and was able to be released and attached to platelet surface upon platelet activation. Purified Angptl2 inhibited agonists-induced human platelet aggregation and spreading on immobilized Fg. Angptl2 inhibited CRP-induced phosphorylation of LAT, SLP76 and PLCγ2, and spreading-driven phosphorylation of FAK Y397 and integrin β3 Y785 in platelet.

Conclusions:
These results revealed that PIRB and its ligand Angptl2 attenuated platelet activation by suppression of GPVI-mediated and integrin αIIbβ3 mediated outside-in signaling. Therefore, LILRB2 and LILRB3 may prove to be the new promising antithrombotic targets.
Abstract 393: Perioperative Cardiovascular Events and Long-Term Mortality Following Orthopedic Surgery
Authors: Oberweis Brandon S , Nukala Swetha , Rosenberg Andrew , Guo Yu , Olivieri Patrick , Bring Rachel , Stuchin Steven , Radford Martha J , Berger Jeffrey S
Published: March 2022

Background:
Adverse cardiovascular events are a major cause of morbidity and mortality in the perioperative period, however data on the association with long-term mortality are lacking. We therefore sought to investigate the long-term prognostic value of cardiovascular events and myocardial necrosis following orthopedic surgery.

Methods:
We performed a long-term follow-up study of 3,082 consecutive subjects undergoing hip, knee, and spine surgery between November 1, 2008 and December 31, 2009. ICD-9 coding was used to ascertain patient characteristics. Perioperative complications of interest were myocardial necrosis (troponin level greater than the 99
th
percentile) and coded myocardial infarction (MI) as defined by ICD-9 coding. Social Security Death Index was used to assess mortality and date of death at a mean follow-up of 3.0 ± 0.5 years. A logistic regression model was used to identify independent predictors of long-term mortality.

Results:
Of the 3,082 subjects, the mean age was 60.8 ± 13.3 years, 41% were male, and 65% were Caucasian. Myocardial infarction occurred in 20 (0.7%) subjects, and among 1062 with post-operatively troponin measured, myocardial necrosis occurred in 179 (16.9%) subjects. The overall incidence of death at a mean follow-up of 3.0 ± 0.5 years was 3.6%. Following multivariable logistic regression, baseline demographics associated with long-term mortality were increasing age (HR 1.04, 95% CI 1.02-1.05, P<0.0001), male sex (HR 1.53, 95% CI 1.04-2.24, P=0.03), emergent or urgent surgery (HR 4.46, 95% CI 2.84-6.99, P<0.0001), cancer (HR 16.61, 95% CI 10.52-26.24, P<0.0001), and diabetes (HR 1.62, 95% CI 1.03-2.54). Both myocardial necrosis (HR 1.84, 95% CI 1.11-3.04, P=0.02) and coded MI (HR 5.11, 95% CI 2.26-11.58, P<0.0001) were independent predictors of long-term mortality.

Conclusions:
Perioperative troponin elevation and coded MI were independent predictors of long-term mortality. This raises the question of whether troponin should be routinely checked in the perioperative setting and whether treating patients found to have myocardial necrosis would attenuate the increased risk of long-term mortality.
Abstract 395: Identification and Characterization of a Novel Mutation in the Low-Density Lipoprotein Receptor Gene in an Omani Arab Family With Familial Hypercholesterolemia
Authors: Banerjee Yajnavalka
Published: March 2022

Autosomal dominant familial hypercholesterolemia (FH) is caused due to mutations in the low-density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9) and apolipoprotein B (ApoB) genes. Little is known regarding its prevalence in the Arab population, which not only exhibits relative genetic homogeneity, but also has a lengthy tradition of consanguinity.

In this study we screened for mutations in FH associated gene in an Omani Arab family, where FH was detected in the index patients according to the Simon Broome criteria. Effect of the identified mutations on different protein structural levels was investigated in silico.

The index patient (9-year-old female) was presented to the lipid clinic with eye xanthelasmata and thickening of both Achilles tendons, and showed off treatment total cholesterol of 896 mg/dL (23.2 mmol/L), low-density lipoprotein cholesterol (LDL-C) of 853 mg/dL (22.1 mmol/L), APOB of 4.5 g/L, triglyceride of 71 mg/dL (0.8 mmol/L), and high-density lipoprotein cholesterol of 0.74 mmol/L. She was treated with a combination of rosuvastatin/ezetimibe and LDL apheresis.

Genetic analysis of the LDLR gene showed a homozygous frame-shift deletion mutation (272delG) at exon 3, which truncates the protein after 90 amino acid residues (versus ~840 amino acids in the wild-type). The mode of inheritance of this novel mutation was also studied to construct a family tree. Additionally it was also confirmed that FH in this family is due to mutation only in the LDLR and not PCSK9 and ApoB genes. Further, the effect of the mutation was also appraised in silico on the tertiary structure of LDLR. A model of the mutant LDLR was constructed using the coordinates of the wild-type LDLR extracellular domain. Based on the model, we present a mechanistic justification behind the observed detrimental effect of the mutation on LDL-C levels.

References:
1. Al-Hinai,A.T. et al. First case report of familial hypercholesterolemia in an Omani family due to novel mutation in the low-density lipoprotein receptor gene. Angiology 64, 287-292 (2013).

2. Al-Rasadi,K. et al. Low-Density Lipoprotein Receptor Gene Mutation Analysis and Structure-Function Correlation in an Omani Arab Family With Familial Hypercholesterolemia. Angiology(2013).
Abstract 4: Apolipoprotein A-IV Confers Protection Against Oxidant Stress and Adipose Tissue Inflammation in Mouse Models of Obesity
Authors: Simon Trang , Weinberg Richard B
Published: March 2022

Background:
Increased lipid oxidation may initiate the chronic inflammatory state which characterizes adipose tissue in obesity. Apo A-IV is a potent in vitro antioxidant, and has been reported to confer protection against multiple inflammatory stimuli in vivo. Apo A-IV may be an especially effective antioxidant in peripheral tissues, as its labile affinity for lipoproteins results in relatively high concentrations in interstitial fluid.

Hypothesis:
Apo A-IV attenuates lipid peroxidation and adipose tissue inflammation in mouse models of obesity.

Methods:
Double heterozygote A4+/- x Ob+/- mice on a C57BL6 background were bred to generate wild type (WT), apo A-IV knockout (A4KO), Ob/Ob, and double knockout (DKO) mice. Groups of age-matched male littermates were fed pro-inflammatory high fat (HF, 42% fat, 0.2% cholesterol) or high fat/high cholesterol (HFHC, 38% fat, 1.25% cholesterol) diets; fasting plasma apo A-IV levels were measured by immunoblotting; plasma total F2 isoprostane levels (a biomarker of global lipid peroxidation) were measured by GC-MS; and expression of genes for pro-inflammatory mediators in adipose tissue was measured in WT and A4KO mice by RT-PCR.

Results:
Baseline plasma apo A-IV levels were higher in the Ob/Ob mice, but increased ~2-fold after 8 weeks on the HF diet. After 12 weeks on the HFHC diet, plasma total F2 isoprostane levels were elevated in Ob/Ob mice, but were highest in DKO mice, indicating that free radical lipid peroxidation in peripheral tissues was increased in the absence of intestinal apo A-IV synthesis. All mice gained weight, but DKO mice gained less than Ob/Ob mice. Adipose tissue expression of the genes for MCP1, IL6, and IL1b was elevated 2-3 fold in A4KO mice.

Conclusions:
These data indicate that plasma apo A-IV levels are chronically elevated by a high fat intake, and that apo A-IV expression attenuates the pro-oxidant/pro-inflammatory state in diet-induced and genetic obesity, which may thereby modulate energy balance.
Abstract 406: Adipose-Specific Deletion of SEIPIN/BSCL2 Results in Progressive Lipodystrophy
Authors: Liu Lu , Jiang Qingqing , Liu George
Published: March 2022

Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) is the most severe form of human lipodystrophy, characterized by an almost complete loss of adipose tissue and severe insulin resistance. BSCL2 is caused by loss-of-function mutations in the BSCL2/SEIPIN gene, which is upregulated during adipogenesis and abundantly expressed in the adipose tissue. The physiological function of SEIPIN in mature adipocytes, however, remains to be elucidated. Here, we generated adipose-specific Seipin knock-out mice (ASKO mice), which exhibit adipocyte hypertrophy with enlarged lipid droplets, reduced lipolysis, adipose tissue inflammation, progressive loss of both white and brown adipose tissue, insulin resistance and hepatic steatosis. Lipidomic and microarray analyses revealed accumulation/imbalance of lipid species including ceramides in ASKO adipose tissue, as well as increased endoplasmic reticulum stress. Interestingly, the ASKO mice almost completely phenocopy the fat-specific Pparγ knock-out mice. Rosiglitazone treatment significantly improved a number of metabolic parameters of the ASKO mice, including insulin sensitivity. Our results therefore demonstrate a critical role of SEIPIN in maintaining lipid homeostasis and the health of adipocytes, and reveal an intimate relationship between SEIPIN and PPARγ.
Abstract 411: Effect of Sitagliptin Therapy on Triglyceride-Rich Lipoprotein Kinetics in Patients With Type 2 Diabetes
Authors: Tremblay André J , Lamarche Benoît , Kelly Isabelle , Charest Amélie , Lepine Marie-Claude , Droit Arnaud , Couture Patrick
Published: March 2022

Postprandial hyperlipemia is a key metabolic feature of insulin-resistant states and an important risk factor for cardiovascular disease, due to the accumulation of atherogenic triglyceride (TG)-rich lipoproteins (TRL) from both hepatic and intestinal origin. Sitagliptin is a selective inhibitor of dipeptidyl peptidase-4 that has been shown to reduce fasting and postprandial glucose levels in patients with type 2 diabetes (T2D) presumably through incretin hormone-mediated improvements in islet function. The objective of the present study was to investigate the effects of sitagliptin therapy on the kinetics of TRL apolipoprotein (apo) B-48, VLDL apoB-100, apoE and apoC-III in patients with type 2 diabetes. Twenty two subjects with T2D (18 men and 4 postmenopausal women, mean age of 58.2±3.8 y) were recruited in this double-blind crossover study using sitagliptin 100 mg/d or placebo for a 6-week period each, with a 4-week washout period between the two phases. At the end of each phase of treatment, the in vivo kinetics of the different apolipoproteins were assessed using a primed-constant infusion of L-[5,5,5-D3]leucine for 12 h with the participants in a constantly fed state. Sitagliptin therapy vs. placebo significantly reduced concentrations of fasting plasma TG (-15.4%, 0.03), apoB-48 (-16.3%, P=0.03), free fatty acids (-9.5%, P= 0.04), HbA1C (placebo: 7.0%±0.8 vs sitagliptin: 6.6%±0.7, P<0.0001), and plasma glucose (-13.5%, P=0.001) without any significant effect on insulin level. Treatment with sitagliptin significantly reduced the pool size of TRL apoB-48 by -20.8% (P=0.03), due to a reduction in the production rate of these particles (-16.0%, P=0.03). VLDL apoB-100 pool size was also significantly decreased by sitagliptin therapy (-9.3%, P=0.03), mainly due to a reduction in the hepatic secretion of these lipoproteins (-9.2%, P=0.06). Finally, sitagliptin treatment had no effect on VLDL apoC-III and apoE levels, but reduced both fractional catabolic and production rates of VLDL apoE (-10.6%, P=0.05 and -12.7%, P=0.04). In conclusion, treatment with sitagliptin for 6 weeks reduces TG-rich apoB-containing lipoproteins levels through a reduction in the synthesis of these particles in patients with T2D.
Abstract 413: Metabolic Channeling from the Insulin Receptor to a Novel Class II Phosphatidylinositol 3’-Kinase and Then to the Threonine-308 Site on AKT Drives Overactive de Novo Lipogenesis in the Obese Liver
Authors: Wu Xiangdong , Williams Kevin Jon
Published: March 2022

Patients with the atherometabolic syndrome develop fatty liver and dysglycemia. Both arise from pathway-
se
lective
i
nsulin
r
esistance and
r
esponsiveness (SEIRR): insulin still drives hepatic
de-novo
lipogenesis, but controls glucose poorly. We reported that injection of insulin into obese
db/db
mice fails to inactivate hepatic PTEN, a crucial action of NOX4. Insulin-stimulated
db/db
livers produce an unusual monophosphorylated form of AKT at Thr308 (pT308-AKT) with only weak phosphorylation at Ser473. Importantly, pT308-AKT activates downstream lipogenic pathways, but fails to phosphorylate FOXO1 or regulate glucose handling. All of these features are recapitulated in cultured hepatocytes by disrupting NOX4.

Any signal from insulin to AKT in the presence of active PTEN is a surprise. Canonically, insulin activates class I PI3 kinases to generate PtdIns(3,4,5)P
3
. This lipid binds pleckstrin homology (PH) domains in AKT and in the two enzymes that phosphorylate AKT (PDPK1 at Thr308 and mTORC2 at Ser473). Persistent PTEN consumes PtdIns(3,4,5)P
3
, blocking this pathway.

To explain insulin-stimulated generation of pT308-AKT, we hypothesized a role for the liver-specific class II PI3K, PI3K-C2γ. Insulin stimulates PI3K-C2γ to generate PtdIns(3,4)P
2
, a lipid that binds PH domains but is a poor substrate for destruction by PTEN. Knock-down of PI3K-C2γ in NOX4-deficient hepatocytes blocked insulin-stimulated formation of pT308-AKT and activation of downstream lipogenic targets.

Here, we explored why PI3K-C2γ activates phosphorylation of AKT at Thr308 over Ser473, even though PDPK1 and mTORC2 both have PH domains. Co-immunoprecipitations revealed a striking difference: PI3K-C2γ robustly associates with PDPK1, but not with SIN1, the component of mTORC2 that contains the PH domain.

Our results demonstrate a parallel, noncanonical signaling pathway in hepatocytes, in which the insulin receptor activates PI3K-C2γ, to generate PtdIns(3,4)P
2
. Because PI3K-C2γ closely associates with PDPK1, not mTORC2, it selectively triggers AKT phosphorylation at Thr308, not Ser473. The result is SEIRR: continued stimulation of hepatic lipogenesis, but a failure of glucose control.
This is the clinical picture of the atherometabolic syndrome.
Abstract 416: IDL-Apolipoprotein E Isoform Navigates Human Coronary Artery Tone
Authors: Mockenhaupt Felicitas , Winkler Karl , Ermilov Eugeny , Hetzer Roland , Siegel Gunter
Published: March 2022

Background:
Apolipoprotein E4/E4 is the most significant risk factor known to cause Alzheimer dementia [1], but also crucial in atherogenesis as pathological correlative of coronary heart disease. The present investigation elucidates the role of IDLapoE isoforms in flow-dependent vascular reactivity.

Methods:
Isometric tension, membrane potential and cyclic nucleotide concentrations were measured in segments of human coronary arteries from heart transplantation.

Results:
The results of the mechanical recording are summarized in the Table. Upon a basal flow rate of 3 mL/min, the initial tension showed a graded course in normal preparations: cooled IDL (10 mg/dL) enhanced mechanical force (+0.8%), IDLapoE4/E4 (+9.5%) and IDLapoE0/E0 (from an apoE ‘knockout man’) (+19.1%) as compared to the controls in Krebs solution. Flow-dependent relaxation was impaired by 20% in apoE4/E4 solution, whereas apoE0/E0 caused a flow-dependent contraction by 85%. Endothelium-denuded segments had basically a higher tone on grounds of an impaired NO release. Membrane potentials reflected tension: hyperpolarization of the VSMCs preceded relaxation, depolarization preceded contraction. For the first time in literature, we report on a chemomechanical coupling curve.

Conclusion:
IDLapoE4/E4 impaired flow-dependent vasodilatation and was only exceeded in its deleterious effect by vasoconstrictive IDLapoE0/E0. There is growing evidence that binding of apoE4/E4 and apoE0/E0 to the flow sensor proteoheparan sulfate leads to nanoplaque build up [2] at the endothelium - blood interface and thus impairs vascular reactivity.

References:
[1] Malmsten et al., JCIS 276 (2004) 503-506; [2] Siegel et al., ATH 144 (1999) 59-67.
Abstract 419: StarD5: Role in a Novel Pathway of Intracellular Cholesterol Transport
Authors: Fortes Carolina , Gil Gregorio , Pandak William M , Rodriguez-Agudo Daniel
Published: March 2022

Introduction:
How cholesterol is moved within the cell via a non-vesicular means and is mobilized against a gradient to the plasma membrane has never been clearly understood. StarD5, a member of the StarD4 subfamily of START (steroidogenic acuteregulatory lipid transfer) domain proteins, is able to bind cholesterol and can be induced by ER stress. Its function, however, has remained a mystery. The objective of this study was to attempt to define the role of StarD5 in intracellular cholesterol metabolism by following its response to ER stress.

Methods:
StarD5 knockdown cells were obtained by using siRNA, while infection with an adenovirus encoding StarD5 was used to selectively overexpress the protein. Immunoblots were performed to determine knockdown and overexpression of StarD5. Filipin staining and immunocytochemistry were used to determine the localization of free cholesterol within the cells. Apoptosis during ER stress was determined by staining cells with Alexa Fluor-488 Annexin V.

Results:
Overexpression of StarD5 in THP-1 macrophages led to a rapid redistribution of StarD5 and cholesterol from the Golgi/ER to the plasma membrane. StarD5 knockdown led to a marked increase in free cholesterol within the Golgi and ER in 3T3-L1 cells. Furthermore, thapsigargin-induced apoptosis (which leads to an abnormal accumulation of cholesterol in the ER) was markedly increased with StarD5 knockdown.

Conclusion:
StarD5 has the ability to respond to different stresses with a directed movement, mobilizing cholesterol between the ER/Golgi to the plasma membrane in order to establish a needed cholesterol membrane concentration gradient amongst intracellular organelles. These findings demonstrate a previously unexplored pathway of intracellular cholesterol transport whose baseline function is induced by ER-stress; likely as a means to stabilize the cell during periods of stress by mobilizing excess cholesterol from the ER and stabilizing the plasma membrane through cholesterol induced changes in permeability and fluidity.
Abstract 422: The Lipid Droplet Associated Protein CIDEC/Fsp27 is Regulated by PPARα and Prevents Hepatic Triglyceride Utilization
Authors: Langhi Cedric , Baldan Angel
Published: March 2022

The cell death-inducing DFFA-like effector c (CIDEC; known as FSP27 or fat-specific protein 27 in rodents) is a lipid droplet (LD)-associated protein that is mainly expressed in white adipose tissue, where it promotes triglyceride storage. Fsp27 levels are undetectable in the livers of fed mice, but rise during fasting or under hepatosteatosis in both obese mice and patients. The Peroxisome Proliferator Activated Receptor α (PPARα) is a nuclear receptor that controls the fatty acid oxidative pathway. Hepatic PPARα signaling is activated physiologically during fasting, and pharmacologically by fibrates, a class of hypotriglyceridemic drugs. Here we show that CIDEC/Fsp27 is induced in primary hepatocytes and in the liver by synthetic PPARα agonists, and promoter reporter and ChIP assays identified CIDEC/Fsp27 as a conserved, direct PPARα target. Importantly, adenoviral-mediated shRNA silencing of hepatic Fsp27 resulted in a strong reduction of fasting-induced hepatosteatosis. Likewise, in vivo shRNA against Fsp27 resulted in decreased hepatic triglyceride contents in mice fed a high-fat diet, and in mice dosed with a PPARα agonist. Further studies in mouse primary hepatocytes showed that loss of Fsp27 activity enhanced PPARα-stimulated fatty acid oxidation. Collectively, our results highlight the physiological role of CIDEC/Fsp27 in hepatic triglyceride homeostasis. Additionally, these data suggest that patients taking fibrates likely have elevated levels of hepatic CIDEC, which may limit the efficient catabolism of hepatic triglycerides.
Abstract 423: A Proatherosclerotic Environment Impacts the Function and Cellular Metabolism in Cardiac Mitochondria
Authors: Rocha Cynthia , Scheede-Bergdahl Celena , Whitesell Thomas , Bergdahl Andreas
Published: March 2022

Apolipoprotein E (ApoE) is an important plasma lipoprotein, able to suppress atherosclerosis by promoting uptake of circulating cholesterol and lipid into the liver. A deficiency of ApoE (ApoE-/-) leads to fatty acid accumulation in the plasma and subsequent increase in reactive oxygen species generation. Mitochondria in cardiomyocytes, due to the nature of the tissue, rely primarily on oxidative pathways for energy production. How this altered lipid metabolism affects cardiac mitochondria, considering the heart relies predominantly on fats as its primary energy source, has yet to be elucidated. The aim of this study is to determine the influence of altered lipid metabolism, resulting from ApoE-/- on cardiac mitochondrial oxidative capacity.

We assessed the hypothesis that cardiac mitochondria would be greatly influenced by a lack of ApoE, thus affecting substrate and energy metabolism, as well as oxygen consumption compared to controls.

2-month-old ApoE-/- mice (n=8) were compared to age-matched C57Bl/6 mice (n=8). All animals were fed a standard chow and had access to water ad libitum. Left ventricular cardiac tissue samples were permeabilized and exposed to a series of titrated substrates and inhibitors that acted on the mitochondrial respiratory complexes. High-resolution respirometry was used to determine mitochondrial oxidative capacity. Citrate synthase activity, immunoblotting and immunofluorescence were also performed. Statistical comparisons were drawn using a two-tailed Student’s t-test. For statistical evaluations, p < 0.05 was considered significant.

The results revealed a decreased basal state 2 respiration, a decreased mitochondrial leak and a higher respiratory control ratio in the young ApoE-/- group compared to controls. Maximal state 3 respiration was similar in both groups.

In conclusion, these findings indicate that ApoE deficiency seems to positively affect the coupling between oxidation and phosphorylation within cardiac mitochondria in young animals. Our results suggest that ApoE-/- actually benefits cardiac metabolism, at least in the early stages of accelerated lipid metabolism. More work is needed to determine if these observations remain as the animals age or whether these effects are time dependent.
Abstract 432: Modification of Lipid Spectrum and Skeletal Muscle Transcriptome Due to Pharmacogenetic Interaction of Retinoic Acid With Rat Chromosome 8
Authors: Krupkova Michaela , Janku Michaela , Liska Frantisek , Sedova Lucie , Hodulova Miloslava , Krenova Drahomira , Kren Vladimir , Seda Ondrej
Published: March 2022

Background:
Polydactylous rat strain PD/Cub and spontaneously hypertensive rat SHR are two established rodent models of human metabolic syndrome. In the process of positional cloning of apparently pleiotropic locus on rat chromosome 8 affecting major features of metabolic syndrome including dyslipidemia we have derived the minimal congenic SHR.PD-(D8Rat42-D8Arb23)/Cub (SHR-Lx) strain carrying only 7 genes of PD origin on SHR background.

Design:
Adult male rats of SHR and SHR-Lx strains were fed standard diet (STD) and subsequently treated with retinoic acid (atRA, 15 mg/kg) via oral gavage for 16 days, while still on STD. We contrasted metabolic profiles (incl. oral glucose tolerance test, free fatty acids, triglyceride and cholesterol in 20 lipoprotein fractions) between SHR and SHR-Lx under conditions of standard diet and standard diet + RA administration. We performed transcriptomic analysis of muscle tissue (m. soleus) in all groups using Affymetrix GeneChip Rat Gene 2.0 ST Arrays followed by Ingenuity Pathway Analysis and real-time PCR validation.

Results:
: In response to RA, SHR-Lx reacted with substantially greater rise in TG and C concentrations throughout the lipoprotein spectrum (two-way ANOVA strain * RA interaction significant for C content in chylomicrons(CM), VLDL and LDL as well as total, CM and HDL-TG). According to our modeling of metabolic and signalization pathways using differentially expressed genes we have identified a network with major nodes (Sirt3, Il1b, Cpt1b, Gria2, Pparg, Rdh11) likely to the observed metabolic differences.

Conclusions:
We demonstrated that pharmacogenetic interaction of retinoic acid with a 7- gene region of rat chromosome 8 affects the distribution of cholesterol and triglycerides into the lipoprotein fractions along with other features of metabolic syndrome. Changed expression of network nodes Pparg, Il1b, Sirt3 and Cpt1b appears as central for observed interaction.
Abstract 436: Macrophage-Specific Cholesterol Efflux Capacity as a Biomarker for High-Density Lipoprotein Function: Comparison of Isotopic Versus Fluorometric Methods for High-Throughput Assessment
Authors: Rohatgi Anand , Givens Edward G , Khera Amit , Ayers Colby R , Shaul Philip W , de Lemos James A
Published: March 2022

Background:
Cholesterol efflux capacity (CEC) is emerging as a novel cardiovascular biomarker of HDL function. To facilitate large-scale human studies, we analyzed analytical performance of CEC and compared a high-throughput fluorometric method with the standard isotopic method.

Methods:
CEC was assessed in J774 cells with either
3
H-cholesterol (isotopic) or BODIPY-cholesterol (fluorometric) to 2.8% apo B-depleted plasma over 4h. Three cohorts were studied: 22 healthy men and women, 38 patients with premature coronary heart disease (CHD) and 196 subjects selected from the Dallas Heart Study (DHS), a population-based study of Dallas county residents. All samples were stored greater than 1 year prior to measurement.

Results:
In healthy subjects (A-B), CEC by both methods did not show diurnal or weekly variation or changes with prandial status, or one freeze/thaw cycle. However, storage at -20
o
C resulted in lower CEC compared to storage at -80
o
C (p=0.02). CEC by both methods was similarly decreased in patients with CHD compared to healthy controls (p=0.06). In 212 DHS subjects, CEC for the two methods were correlated with each other (r=0.54, p<0.0001) but had differential correlations with HDL-C and HDL-P (figure).

Conclusions:
CEC to apo B-depleted human plasma is not influenced by timing of sampling or storage conditions, with the exception of storage at -20
o
C. A 96-well high-throughput fluorometric method using BODIPY-cholesterol exhibited similar performance characteristics as the standard isotopic method with differing correlations with HDL composition and may therefore be advantageous in large scale human studies.
Abstract 44: Tissue-Protective Effects of an Erythropoietin Derivative in in vitro and in vivo Models of Skeletal Muscle Ischemia
Authors: Yu Rebekah Sian Hwee , Baker Daryll , Abraham David , Tsui Janice C S
Published: March 2022

Introduction:
A third of patients with critical limb ischemia (CLI) eventually require amputation. In spite of clinically successful revascularization patients rarely return to their pre-morbid status, and often fail to notice an improvement in their functional outcomes, which may be due to an underlying musculopathy. Non-hematopoietic EPO-derivatives have been designed to retain only tissue-protective functions of EPO. We hypothesized that ARA-290 (EPO-derivative) may have tissue-protective potential that would represent a novel therapeutic adjunct in patients with CLI.

Methods:
The effect of ARA-290 in mediating cytoprotection was assessed firstly in vitro using skeletal myoblasts isolated from CLI and control donors, and a model of simulated ischemia. Characterization of CLI myoblasts was also performed, to asses their contractile, migratory and proliferative ability. Subsequently, an in vivo murine model of hindlimb ischemia, which recapitulates the muscular pathology observed in CLI patients, was used to assess the potential of ARA-290 to improve functional, histological and perfusion outcomes.

Results:
Skeletal myoblasts were successfully isolated from CLI patients for the first time. CLI myoblasts and myotubes exhibited increased proliferative capacity but reduced migratory and contractile function and importantly a reduced susceptibility to a second ischemic-insult compared with control myoblasts and myotubes. ARA-290 treatment led to significant improvements in both CLI and Control myoblasts and myotube function and survival via the JAK2/STAT3, PI3k/Akt and NFκB signalling pathways. In vivo, animals treated with ARA-290 demonstrated improved functional, histological and perfusion outcomes compared to vehicle-control treated animals.

Conclusion:
These studies demonstrate the potential of ARA-290 to protect tissues and cells from ischemic-injury and encourages the development of novel pharmacological therapies for use in patients with “no option” CLI or severe functional deficit.
Abstract 441: Epicatechin Attenuates Atherosclerosis and Exerts Anti-inflammatory Effects on Diet-Induced Human CRP and NfκB in vivo
Authors: Morrison Martine , Van der Hoorn José W , Van der Heijden Roel , Heeringa Peter , Kaijzel Eric , Verschuren Lars , Blomhoff Rune , Kooistra Teake , Kleemann Robert
Published: March 2022

Objective:
Previous studies investigating flavanol-rich foods provided indications for potential cardioprotective effects of these foods, but the effects of individual flavanols remain unclear. We investigated whether the flavanol epicatechin can reduce diet-induced atherosclerosis, with particular emphasis on the cardiovascular risk factors dyslipidaemia and inflammation.

Methods:
ApoE*3Leiden mice were fed a cholesterol-containing atherogenic diet with or without epicatechin (0.1% w/w) to study effects on early- and late-stage atherosclerosis (8w and 20w).
In vivo
effects of epicatechin on diet-induced inflammation were studied in human-CRP transgenic mice and NFκB-luciferase reporter mice.

Results:
Epicatechin attenuated atherosclerotic lesion area in ApoE*3Leiden mice by 27%, without affecting plasma lipids. This anti-atherogenic effect of epicatechin was specific to the severe lesion types, with no effect on mild lesions.

Epicatechin mitigated diet-induced increases in plasma SAA (in ApoE*3-Leiden mice) and plasma human-CRP (in human-CRP transgenic mice). Microarray analysis of aortic gene expression revealed an attenuating effect of epicatechin on several diet-induced pro-atherogenic inflammatory processes in the aorta (e.g. chemotaxis of cells, matrix remodelling), regulated by NFκB. These findings were confirmed immunohistochemically by reduced lesional neutrophil content in HCE, and by inhibition of diet-induced NFκB activity in epicatechin-treated NFκB-luciferase reporter mice.

Conclusion:
Epicatechin attenuates development of atherosclerosis and impairs lesion progression from mild to severe lesions in absence of an effect on dyslipidaemia. The observed reduction of circulating inflammatory risk factors by epicatechin (e.g. SAA, human-CRP), as well as its local anti-inflammatory activity in the vessel wall, provide a rationale for epicatechin’s anti-atherogenic effects.
Abstract 452: Signal Transducer and Activator of Transcription 4 Deficiency Improves the Metabolic Phenotype and Skeletal Muscle Insulin Signaling in LDLr-/- Mice on Diabetogenic High-Cholesterol Diet
Authors: Ma Kaiwen , Hatcher Margaret , Glenn Lindsey , Tagavie-Moghadan Paresa , Nadler Jerry , Dobrian Anca D
Published: March 2022

Insulin resistance and type2 diabetes are associated with increased incidence of cardiovascular disease. About 80% of the subjects with type2 diabetes die due to cardiovascular complications. We previously showed that STAT4 deficiency improves insulin resistance in C57Bl6 mice on high fat diet and STAT4-/-ApoE-/- mice develop significantly lower atherosclerosis compared to ApoE-/- controls. The goal of this project was to determine the effect of STAT4 deficiency in a combined model of atherosclerosis and insulin resistance. To this purpose we generated STAT4-/-LDLr-/- mice and fed them either a chow diet or a high carbohydrate (36.6%), high cholesterol (0.15%) (DDC) diet for 16 weeks (n=7-10). The LDLr-/- on DDC diet developed glucose intolerance and insulin resistance as well as atherosclerosis and hypercholesterolemia compared to chow fed controls. STAT4-/-LDLr-/- mice on DDC diet, although hypercholesterolemic had significantly reduced atherosclerotic plaques by en face staining (p<0.05). Also STAT4 deficient mice on DDC diet had significantly lower area under curve for both the insulin and glucose tolerance tests (p<0.01), as well as reduced fasted glucose (p<0.05) compared to LDLr-/- mice despite similar body weights. Glucose stimulated insulin secretion in isolated pancreatic islets was blunted in LDLr-/- mice on DDC compared to chow controls and STAT4 deficiency restored the insulin secretion in islets from DDC fed mice at levels comparable to chow controls. In vivo insulin challenge showed reduced activation of IR, IRS-1 and Akt in LDLr-/- mice on DDC compared to chow controls in adipose tissue and skeletal muscle. STAT4-/-LDLr-/- on DDC diet had significantly improved activation of IRS-1 and Akt in skeletal muscle but not in adipose tissue. IR activation was constitutively higher in in STAT4-/-LDLr-/- mice on DDC compared to chow and did not change after insulin stimulation suggesting that STAT4 deficiency restores insulin signaling via a post-receptor mechanism. In conclusion, STAT4 deficiency reduces insulin resistance and improves glucose intolerance primarily by improving insulin signaling in skeletal muscle and by increasing islet insulin secretion. This may explain in part the athero-protective phenotype in STAT4-/-LDLr mice.