bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 147: Plasma Factor XIII Binding to Cold-stored Platelets Results in Increased Fibrin Crosslinking and Clot Strength
Authors: Nair Prajeeda M , Pandya Shaunak G , Dallo Shatha F , Reddoch Kristin M , Montgomery Robbie K , Pidcoke Heather F , Cap Andrew P , Ramasubramanian Anand K
Published: July 2021

Currently, platelets (PLTs) stored at room temperature (RT) for 5-7 days with gentle agitation are exclusively used for transfusion although FDA recently clarified that apheresis PLTs stored at 4°C for up to 72 hours may be used for treating active hemorrhage. We have demonstrated that cold (4C) storage of PLT is an attractive alternative to RT storage since it better preserves the PLT metabolic reserves,
in vitro
responses to agonists of activation, aggregation and physiologic inhibitors, as well as adhesion to thrombogenic surfaces. In this study, we tested the hypothesis that 4C-stored PLT will form clots with mechanical strength superior to those from RT-stored PLT due to higher hemostatic potential. From rheological measurements, we observed that the clots formed from 5 day 4C-stored PLTs are significantly stiffer (elastic modulus) and stronger (critical stress) than those formed from RT-stored PLT but comparable to fresh PLT (Fig. A). We also observed from ultrastructural microscopy that the fibrin fibers in clots from cold-stored PLT were thinner with more branch points than those from RT-stored PLTs, indicating the presence of increased crosslinks (Fig. B, C). Finally, molecular analysis revealed an increase FXIII transglutaminase activity due to the binding of plasma FXIII/fibrinogen to the surface of 4C-stored PLTs (Fig D).In conclusion, we have shown that cold-induced plasma FXIII binding to PLT surface result in increased fibrin crosslinking and enhanced clot strength. Our data, together with the benefit of reduced risk of late thrombosis due to their rapid clearance
in vivo
, underscores the consideration of 4C-stored PLT for acute response to hemorrhage.
Figure 1
(A) RT-stored platelets form clots with less stiffness (n=5); (B) Representative SEM images of clots (n=3) (C) Clots from 4C-stored platelets have higher cross-linking density (n=5) (D) In FXIII-deficient plasma, 4C-stored platelets form clots with higher stiffness than fresh platelets (n=4)
Abstract 148: Difference in Neointimal Coverage Pattern Between Zotarolimus Eluting Stents and Everolimus Eluting Stents Using Optical Coherence Tomography: A Meta-Analysis
Authors: Yarlagadda Vivek , Agasthi Pradyumna , Chaudhary Aneese , Aloor Sivakanth , Chang Eric , Lee Kwan , Kumar Gautam , Sachdeva Rajesh
Published: July 2021

Background:
Late stent thrombosis in drug eluting stents (DES) is attributed to poor endothelialization of struts. Second generation DES have shown superior safety profile and efficacy in comparison to first generation DES. We conducted a meta-analysis to compare the neointimal coverage patterns between Zotarolimus eluting stents (ZES) and Everolimus eluting stents (EES) using optical coherence tomography (OCT).

Methods:
We searched online databases for studies comparing the neointimal thickness (NIT), %malapposed strut per stent (MSS) or % uncovered strut per stent (USS) between ZES and EES using OCT. Studies included inte mmeta-analysis, OCT was performed >6 months after stent deployment. A calculation of weighted standardized mean difference (SMD) in NIT, MSS and USS between ZES and EES groups was calculated. Residual maximum likelihood (REML) metaregression was performed on smokers, diabetes mellitus (DM), hypertension, (HTN) dyslipidemia (DLD), and age and sex covariates.

Results:
A total of 6 studies enrolling 327 patients which met inclusion criteria were included in the meta-analysis. 400 stents including 184 ZES and 216 EES were analyzed by OCT. The median of mean age in years, male sex, smokers, DM, HTN and DLD in the ZES group was 60.7(59.5 - 60.9), 76.7% (71.4 - 77.3), 38.7% (18.2 - 45.8), 36.4% (23.3 - 37.5), 63.6% (60 - 66.6), 66.7% (47.9 - 68.4) versus 62.6(59.8 - 65.3), 63.9% (63.6 - 82.1), 30% (17.2 - 47.2), 31.8% (25 - 36.1), 69.4% (59.1 - 75.9), 70% (48.9 - 70.7) in the EES group respectively. The unweighted median NIT, MSS and USS in the ZES group are 127.5 μm (108.8 - 152.2), 0.7% (0.7 - 0.9), 1.8% (0.2 - 3.5) vs 117.4 μm (108.5 - 128.5), 0.3% (0.2 - 1.3), 2.3% (0.9 - 3.5) in the EES group respectively. The weighted SMD of NIT, MSS and USS in the ZES vs EES groups are -0.22 (95%CI -1.12; 0.67) P = 0.63, -0.51(95% CI -1.78; 0.77), P = 0.44, -1.26 (95%CI -2.83; 0.31). None of the above mentioned covariates accounted for statistical insignificance except age for NIT. As age increases, the SMD increases for NIT and was not significant for MSS and USS.

Conclusion:
No significant difference was noted in the NIT, MSS and USS between both groups. This suggests that the neointimal coverage patterns are similar in ZES and EES groups.
Abstract 149: Phosphatidylserine Promotes Thrombosis in Pediatric CPB Model
Authors: Meyer Andrew D , Raghunath Anjana , Heard Patricia , Kamucheka Robin , Lafleur Crystal , Scherer Michael , Cap Andrew P
Published: July 2021

Over 18,000 children per year receive cardiopulmonary bypass (CPB) surgery. Unfortunately, common CPB-related thrombotic complications continue to result in significant mortality and morbidity. Previous ex-vivo CPB studies using animal blood document an increase in platelet-derived microparticles (PMPs), which are small (0.1-1 micron) membrane vesicles that may be 50 to 100-fold more pro-coagulant than activated platelets. Our hypothesis is that increased duration and magnitude of shear stress in an ex-vivo pediatric CPB circuit increases the generation of PMP expressing pro-thrombotic phosphatidylserine. We constructed an ex-vivo CPB circuit that circulates heparinized human blood from healthy adult volunteers for six hours at pediatric flow rates (
e.g.,
0.3, 0.5, and 0.7 L/min). Our protocol normalizes each run through the circuit to a normal hematocrit, pH, ionized calcium, and an activated clotting time of 180 to 220 sec. An aliquot of static blood controls is maintained in a similar test environment without CPB circuitry. PMP-PS (CD41a+/phosphatidylserine[PS]) concentration and pro-coagulant function are measured in platelet-depleted plasma using high-resolution flow cytometry (BS FACS Canto II with PMT) and STA®-Procoag-PPL. Thrombin generation (
e.g.
, calibrated automated thrombogram) and clot formation (
e.g.
, thromboelastography) further define the coagulation function of pump-produced PMP-PS. At 0.5 L/min the circuit generates an exponential increase in PMP-PS and decreasing PPL clot time compared to static blood control (
p
<0.001, Figure 1, n=4). Platelet count, prothrombin time, and activated partial thromboplastin time do not change over time. Results also document an increase in peak thrombin potential and clot formation that correlate strongly with the increase in PMP-PS. PMP-PS may be a clinically relevant biomarker and therapeutic target to decrease life-threatening CPB surgery coagulation complications.
Abstract 15: Serum Amyloid A is Not Just an HDL-Associated Lipoprotein
Authors: Wilson Patricia G , Thompson Joel C , Shridas Preetha , de Beer Frederick C , Webb Nancy R , Tannock Lisa R
Published: July 2021

Serum Amyloid A (SAA) is traditionally thought to be only found with HDL; however, recently several groups have found that SAA is found on apoB-containing lipoproteins (LDL and VLDL) under some circumstances. The goal of this study was to determine the relative lipoprotein association of SAA. Native human acute phase SAA was isolated from acute phase plasma collected from patients undergoing cardiovascular bypass surgery; the SAA was purified and delipidated. Plasma was collected from a group of healthy, non-obese humans with low levels of SAA (< 2 mg/L) and lipoproteins (VLDL, LDL and HDL) were isolated by density gradient ultracentrifugation. The delipidated SAA (at 2 concentrations) was incubated in vitro with the various lipoprotein preparations then samples were re-isolated by fast protein liquid chromatography and SAA was analyzed by ELISA and immunoblot. When SAA was incubated with any single lipoprotein all of the SAA was found associated with that lipoprotein and none remained in a lipid-free form. When SAA was incubated with a mixture of VLDL, LDL and HDL (based on equal protein and corresponding to concentrations in plasma) a majority of SAA (50-60%) was found on HDL with the remaining SAA found on VLDL and LDL. When SAA first complexed to HDL was added to a mixture of SAA-free LDL and VLDL the majority of SAA remained with the HDL (76-86%) but 5-10% of the SAA was found on each of LDL and VLDL. When SAA first complexed to either apoB-containing lipoprotein was then added to a mixture of HDL and the other apoB-containing lipoprotein most SAA moved to HDL (55-70%) but the remainder was found on the apoB-containing particles. Thus, SAA can move between lipoprotein particles in vitro. To determine if the presence of SAA on apoB-containing lipoproteins had a functional effect we evaluated lipoprotein-proteoglycan binding affinity. Proteoglycan mediated lipoprotein retention in the vessel wall is thought to be one of the key steps in initiation of atherosclerosis. Compared to SAA-free LDL or VLDL, the presence of SAA on apoB-containing lipoproteins caused increased proteoglycan binding affinity. Thus, SAA is not simply an HDL lipoprotein, but SAA can move between lipoprotein particles, and the presence of SAA on apoB particles may increase their atherogenicity.
Abstract 150: Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype
Authors: Cameron Scott J , Mix Doran S , Ture Sara K , Schmidt Rachel , Mohan Amy , Toth Sandra , Stoner Michael C , Field David J , Modjeski Kristina L , Morrell Criag N
Published: July 2021

Background:
An understudied area of platelet biology is that ischemic diseases such as peripheral vascular disease (PVD) can alter the platelet phenotype, and this may lead to unpredictable effects of anti-platelet agents.

Methods:
Isolated murine and human platelets were exposed to a reduced oxygen environment (hypoxia chamber, 5% O
2
). Platelet activation was assessed as mean fluorescence intensity (MFI) of surface p-selectin by flow cytometry, and morphologic changes by confocal microscopy. A murine model of critical limb ischemia (CLI) was used to assess changes in platelet activity in wild-type (WT) and in platelet ERK5
-/-
mice. Limb blood flow was assessed by laser Doppler imaging. Hypoxia was additionally examined in mice by unilateral pneumonectomy. Western blotting was used to assess protein expression.

Results:
In human platelets
ex vivo
at 5% O
2
, platelet activation increased to 5076 MFI±409 from 3548 MFI±187 at 20% O
2
(P=0.005). With the ERK5 inhibitor XMD892, platelet activation at 5% O
2
decreased from 5677 MFI±312 to 3175 MFI±247 (p=0.001). In a murine model of PVD, enhanced platelet activation was sustained for weeks vs. sham, and limb blood flow in platelet ERK5
-/-
mice one week after ischemia was augmented by 26%: 0.61±0.03 vs 0.87±0.03 vs, WT mice (p=0.0002), with 50% less platelet activity. In mice with unilateral pneumonectomy vs. sham surgery, observed hypoxia was accompanied by increased platelet ERK5 activation coincident with enhanced platelet activation. In patients with CLI, augmented platelet activation in spite of aspirin treatment was observed via the thromboxane and the PAR1 receptors, and especially via P2Y
12
receptor.

Conclusions:
Hypoxia and ischemic tissue injury, such as with critical limb ischemia, change the phenotype of the platelet, promoting a pro-thrombotic state that is partly dependent on platelet ERK5. Platelet phenotype and function should be better characterized in ischemic diseases.
Abstract 166: Acceleration of Diabetic Wound Healing With Adipose Derived Stem Cells, Endothelial Differentiated Stem Cells and Topical Conditioned Medium Therapy in a Swine Model
Authors: Irons Robin F , Cahill Kevin W , Rattigan Deviney A , Marcotte Joseph H , Fromer Marc W , Chang Shaohua , Zhang Ping , Behling Kathryn C , Brown Spencer A , Carpenter Jeffrey P , Caputo Francis J
Published: July 2021

Objectives:
The goal of our study is to investigate the effect of adipose derived stem cells (ASCs), endothelial differentiated (ED) ASCs, and various conditioned medium (CM) on wound healing in a diabetic swine model.

Methods:
Diabetes was induced in 4 male Yorkshire pigs via intravenous injection of 150 mg/kg Streptozotocin. Pig ASCs were harvested and cultured in either M199 or EGM-2 medium with 30 mm glucose. Fourteen circular dorsal wounds (5 cm diameter; 5 mm depth) were created. Duplicate wounds were treated with one of 4 cell-based therapies by injection of 5 million (M) ASCs, 10M ASCs, 5M ED ASCs, or 10M ED ASCs on day 0 and half the initial dose on day 15. Wounds assigned to the topical CM therapy were covered with 2 cc of either serum free M199 primed by ASCs or Human Umbilical Vein Endothelial Cells every 3 days. One set of wound was designated as control. Wounds were assessed at day 0, 10, 15, 20 and 28. Animals were sacrificed on day 28. Image J software was used to evaluate percent wound healing.

Results:
On day 10, 15, 20 and 28 there was a significant increase in percent wound closure rates in both cell-based treatments when compared to control (p<0.05,
Figure 1
). All treatments displayed significant increase in wound closure rates on day 28 when compared to control. Histology revealed a decrease in dermal inflammatory cells and improved restoration of normal skin architecture in therapeutic treatment groups when compared to untreated controls.

Conclusions:
Cellular therapy with ASCs, ED ASCs and topical CM accelerate diabetic wound healing in the swine model. Cellular therapy may provide the greatest benefit, as cells may continuously supply growth factors and adapt to the evolving microcellular environment as the wound progresses through the various phases of healing
Abstract 17: Apolipoprotein C-III Promotes Anti-Inflammatory Regulatory T Cell Phenotype Through Modulation of Intestinal Dendritic Cell Activation
Authors: Rodia Cayla N , Kohan Alison B
Published: July 2021

Apolipoprotein C-III (apoC-III) is a cardio-metabolic modulator in multiple tissues. Circulating apoC-III in humans is an independent risk factor for cardiovascular disease (CVD) because apoC-III increases circulating triglyceride (TG) through inhibition of the LDLR in the liver and lipoprotein lipase on the capillary endothelium. In the liver, apoC-III overexpression results in increased synthesis and secretion of more TG-rich VLDL. These actions increase the resident time and amount of atherogenic remnants in circulation. Paradoxically, apoC-III acts in the intestine to inhibit dietary fat absorption, a potentially beneficial action. Using human-apoC-III transgenic mice (hu-apoCIII
tg
mice), which have increased circulating TG compared to C57Bl6/J littermate controls (WT), we have identified an increase in circulating IL-10 at basal conditions. Regulatory T cells (Tregs), a T cell subset that is known to delay atheroprogression, secrete the anti-inflammatory cytokine IL-10. Knowing that the immune system plays a dynamic role in atherogenesis, we asked whether apoC-III is a potential modulator of Tregs during a western diet (WD) challenge. We subjected hu-apoCIII
tg
and WT mice to 12 weeks of a WD and through flow cytometry, found significantly increased circulating and intestinal Tregs in hu-apoCIII
tg
mice compared to WT. Hu-apoCIII
tg
intestinal gene expression showed increases in TGF-β and maintenance of IL-10 and FOXP3, key Treg-related genes, after the WD compared to WT. We hypothesized that apoC-III may act in the intestine to induce tolerance to dietary antigens through dendritic cell (DC) activation and subsequent differentiation of naïve T cells to the Treg phenotype. Using flow cytometry of the lamina propria, we saw increased activation of intestinal DCs, in hu-apoCIII
tg
mice at basal and WD-challenged conditions suggesting that intestinal DCs are influenced by apoC-III overexpression. These results suggest that apoC-III may influence Treg differentiation or proliferation through alteration of intestinal DC activation.

The phenotype we have uncovered in hu-apoC-III
tg
mice suggests a link between apoC-III overexpression, dietary fat absorption, and the immune system, and that this may be protective against inflammation.
Abstract 175: Glycosylated Hemoglobin is Associated With Coronary Plaque Burden in Psoriasis Beyond Cardiovascular Risk Factors
Authors: Chung Jonathan H , Rana Anshuma , Dey Amit K , Rivers Joshua P , Chaturvedi Abhishek , Lerman Joseph B , Rodante Justin A , Harrington Charlotte L , Playford Martin P , Chen Marcus Y , Bluemke David A , Mehta Nehal N
Published: July 2021

Introduction:
Psoriasis (PSO), a chronic inflammatory disease associates with increased metabolic and cardiovascular (CV) risk. Dysglycemia may contribute to the increased risk of CV disease observed in psoriasis. Therefore, the aim of this study was to characterize whether dysglycemia assessed by hemoglobin A1c (HbA1c) is associated with subclinical coronary artery disease in psoriasis.

Hypothesis:
We hypothesized that total burden (TB) of coronary artery plaque and non-calcified burden (NCB) would be associated with HbA1c values in psoriasis independent of cardiovascular risk factors.

Methods:
Consecutive psoriasis patients (n=103) and age and sex matched controls (n=42) underwent CCTA (Toshiba, 320-detector row) for coronary plaque characterization. TB and NCB were quantified using QAngio (Medis, The Netherlands). Fasting blood samples were collected on the same day to get HbA1c values. The relationship of HbA1c with TB and NCB was analyzed using multivariable regression models (STATA 12).

Results:
PSO patients were middle-aged, predominantly male, had a low CV risk by FRS and had mild to moderate skin disease (Table 1). HbA1c levels were elevated in psoriasis compared to controls (P=0.007). Furthermore, total (P=0.02) and non-calcified (P=0.04) burden were elevated in psoriasis as compared to controls. HbA1c directly associated with TB (
β
=0.21; P=0.001) and NCB (
β
=0.18; P=0.004); a relationship which persisted beyond CV risk factors (TB and NCB:
β =0.22;
P=0.01).

Conclusions:
HbA1c was associated with total and non-calcified burden in psoriasis population independent of CV risk factors. These findings underscore the importance of screening for glycemic abnormalities to mitigate CV disease risk in patients with psoriasis.
Abstract 184: Adenylate Cyclase Type 9 Promotes Atherosclerosis in Mice
Authors: Rautureau Yohann , Deschambault Vanessa , Brand Geneviève , Higgins Marie-Eve , Mecteau Mélanie , Uy Kurunradeth , Rivas Daniel , Samami Samaneh , Mayer Gaétan , Rhéaume Eric , Tardif Jean-Claude
Published: July 2021

Polymorphisms in the ADCY9 gene, coding for adenylate cyclase type 9, determine atherosclerotic cardiovascular responses to the CETP inhibitor dalcetrapib in patients. ADCY9 is broadly expressed and involved in various immune cell functions and inflammatory responses. Our objective is to determine the role of ADCY9 in the development of atherosclerosis in the absence of CETP.

We used 8-12 week-old wild-type (WT, n=25) or
Adcy9
-inactivated (
Adcy9
Gt
, n=21) male mice. To induce hypercholesterolemia and atherosclerosis, mice were infected with an adeno-associated virus coding for a gain-of-function mutant of
Pcsk9
(
Pcsk9
D377Y
) and were fed an atherogenic high-cholesterol diet for 16 weeks in absence or presence of dalcetrapib treatment (200 mg/kg/day). Percent atherosclerotic lesion area (PALA) in the whole aorta was quantified using
en face
Oil Red O plaque staining. We used VCAM-1 immunofluorescence staining (n=6 per group) in atherosclerotic lesions of the aortic root to quantify inflammation. We also measured aortic root accumulation of macrophages and vascular smooth muscle cells by immunofluorescence imaging of CD68 and smooth muscle actin (SMA), respectively, in plaque lesions. We also quantified blood leukocytes in normocholesterolemic WT and
Adcy9
Gt
mice by flow cytometry analysis of CD45
+
cells.

In hypercholesterolemic animals,
Adcy9
Gt
mice showed a 60% decrease in atherosclerotic lesions (PALA: 2.7±0.5%) compared to WT mice (6.5±0.7%, P<0.01). Dalcetrapib treatment, in these mice without CETP, did not modify significantly the reduction (63%) of PALA in
Adcy9
Gt
mice (2.9±0.4%) compared to WT (7.6±1.6%, P<0.01). Macrophage content was reduced by 55% from 17.8±2.8% in WT to 7.2%±1.9% in
Adcy9
Gt
mice (P<0.05), while SMA staining was similar. VCAM-1 expression also tended to be decreased, from 5.7±3.3% in WT to 2.0±0.4% in
Adcy9
Gt
mice (P=0.0598). CD45
+
blood leukocytes were reduced by 35.4% in
Adcy9
Gt
compared to WT normocholesterolemic mice (P<0.01).

Adcy9
inactivation in mice (without CETP) results in large reductions of atherosclerosis and plaque macrophage and in decreased VCAM-1 expression. These results support the ADCY9 genotype-dependent clinical effects of dalcetrapib.
Abstract 185: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 in Mice Lowers Asymmetric Dimethylarginine and Improves Vasomotor Function
Authors: Rodionov Roman N , Burdin Dmitri , Brilloff Silke , Todorov Vladimir , Jarzebska Natalia , Martens-Lobenhoffer Jens , Hofmann Anja , Morawietz Henning , Hilgers Karl , Cordasic Nada , Jacobi Johannes , Maas Renke , Chen YingJie , Bode-Böger Stefanie M , Hugo Christian P , Hohenstein Bernd , Weiss Norbert
Published: July 2021

Background:
It has been demonstrated in various studies that ADMA (asymmetric dimethylarginine), an inhibitor of nitric oxide synthase, is associated with the increased risk of cardiovascular diseases. There are two known pathways of ADMA metabolism: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolases (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The second pathway is still poorly understood. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and improvement of vasomotor function.

Methods and Results:
We generated transgenic mice (TG) with ubiquitous overexpression of
AGXT2
under control of the chicken beta actin (CAG) promoter. qPCR and Western Blot were used to confirm the ubiquitous expression of the transgene. HPLC-MS/MS was used to generate biochemical data. Systemic ADMA levels were decreased by 15% (p<0.05) in the TG mice, whereas ADGV plasma levels were six times higher in comparison with wild type animals (p<0.001). Heart and lung of TG animals exhibited 2 times lower tissue ADMA content in comparison with wild type littermates (p<0.05). In further experiments, we crossed the AGXT2 TG mice with DDAH1 KO mice and showed that upregulation of AGXT2 protects DDAH1 KO mice from elevation of plasma ADMA levels and restores endothelium-dependent vasodilation in aortic rings. In the current experiments we are assessing whether AGXT2 overexpression also protects DDAH1 KO mice from hypertension.

Conclusion:
In the current study we demonstrated that upregulation of AGXT2 leads to lowering of ADMA levels and improvement of endothelium-dependent relaxation
in vivo
in the settings of DDAH1 deficiency
.
AGXT2 thereby may be a potential drug target for long-term reduction of systemic ADMA levels in cardiovascular pathologies. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far.
Abstract 186: Cyp8b1 Ablation Prevents Western Diet-Induced Weight Gain and Hepatic Steatosis due to Impaired Fat Absorption
Authors: Bertaggia Enrico , Jensen Kristian K , Castro-Perez Jose , Xu Yimeng , Chan Robin B , Wang Liangsu , Haeusler Rebecca A
Published: July 2021

Bile acids (BAs) are cholesterol derivatives that are well-known for their role in facilitating intestinal lipid absorption. Furthermore, they can regulate glucose and lipid metabolism by activating nuclear and cell surface receptors. Insulin resistance is correlated with alterations in bile acid composition, in particular higher levels of 12α-hydroxylated BAs. These BA species are generated by the enzyme Cyp8b1. We hypothesized that elevated levels of 12α-hydroxylated BAs could be a link between insulin resistance and defects of lipid metabolism. To study the role of Cyp8b1 in the regulation of lipid metabolism, we used Cyp8b1 deficient mice-which are unable to produce 12α-hydroxy BAs-and challenged them with a western type diet. We found that Cyp8b1-/- mice gained less weight compared to controls, which was entirely accounted for by fat mass. Triglyceride and cholesterol accumulation in the liver of Cyp8b1-/- mice were also strongly reduced. We found that these improvements were due to reduced lipid absorption in the intestine of Cyp8b1-/- mice, which could be rescued by replenishing the pool with taurocholic acid. The lipid malabsorption resulted in higher caloric excretion in the feces, due to excess excretion of hydrolyzed dietary lipids. When we fed the mice with a fat-free diet, these differences between genotypes were normalized, confirming the central role of BA composition-not just overall levels-in intestinal lipid absorption and whole-body lipid homeostasis. Based on these findings, it is possible that reducing 12α-hydroxy BAs could be a therapeutic option for the control of obesity and lipid homeostasis.
Abstract 188: Angiopoietin-Like Protein 4 is a HDL Component For HDL Metabolism snd Function in Non-Diabetic Participants and Type-2 Diabetic Patients
Authors: Yang Longyan , Yu Caiguo , Wang Xuhong , Yuan Shasha , Zhang Lijie , Lang Jianan , Zhao Dong , Feng Yingmei
Published: July 2021

Objectives:
Angiopoietin-like protein 4 (angptl4) is a potent lipoprotein lipase inhibitor and present in HDLs in mice and human. Plasma levels of ANGPTL4 were increased in type-2 diabetic (T2DM) patients. However, it is not defined whether ANGPTL4 in HDLs could participate in HDL metabolism and affect its function in T2DM.

Methods:
Non-diabetic (n=201) and T2DM patients (n=185) were analyzed in the study. Angptl4 levels in the circulation and HDLs were quantified by ELISA. HDL components were measured. HDLs were isolated to assess cholesterol efflux or subjected to endothelial lipase (EL)-expressing HEK293 cells to study the kinetic of EL hydrolysis
in vitro
.

Results:
The levels of angptl4 in the plasma and HDLs were 1.7- and 2.0-fold higher in T2DM patients than non-diabetic controls (p<0.0001 for both). When angptl4-containing HDLs were subjected onto HEK 293 cells expressing EL, phospholipid and triglyceride were dramatically hydrolyzed in HDLs with medium- and high-levels of angptl4 when compared with those having lowest angptl4 levels in HDLs in T2DM patients (p<0.05 for phospholipid; p<0.01 for triglyceride), but it did not occur in HDLs isolated from healthy controls (p>0.62 for both).Cholesterol hydrolysis was comparable and independent of angptl4 levels in HDLs in both groups. Multivariate-adjusted analysis demonstrated that per one doubling increase of ANGPTL4 levels in HDLs, the changes amounted to +0.27% cholesterol efflux (p=0.03), +0.06 μg/mL apoA-I (p=0.09) and -9.41 μg/L SAA (p=0.02) in non-diabetic controls. In T2DM patients, the corresponding estimates were -0.06% cholesterol efflux (p=0.10), -0.06 μg/mL apoA-I (p=0.38) and +3.64 μg/L SAA (p=0.72).

Conclusions:
Angptl4 in HDLs protected HDLs from hydrolysis and maintained HDL function in non-diabetic controls. Resulting from increased circulating angptl4 levels in T2DM, angptl4 levels in HDLs were elevated that compromised its inhibitory effect on EL, leading to increased HDL hydrolysis and dysfunction.
Abstract 189: Improved Triglyceride-Rich Lipoprotein Clearance In Human Apolipoprotein A-Ii Knockin Rabbits
Authors: Koike Tomonari , Koike Yui , Yang Dongshan , Song Jun , Xu Jie , Fan Jianglin , Zhang Jifeng , Chen Y. Eugene
Published: July 2021

Apolipoprotein A-II (apoA-II) is a second major apolipoprotein in HDL particles, which account for 15-20 % of total HDL protein in human. Compared with apolipoprotein A-I (apoA-I) which is a most major apolipoproteins in HDL, physiological role of apoA-II is not fully understood. We previously reported that overexpression of apoA-II in rabbits significantly suppressed cholesterol-rich diet induced atherosclerosis. Because rabbits are the animal of naturally deficient in apoA-II, so it becomes a good animal model to study the functions of apoA-II. To elucidate the role of apoA-II independent from apoA-I in HDL on lipid metabolism and atherogenesis, we generated a rabbit model in which the human apoA-II was knocked in to rabbit apoA-I locus by TALEN technology. The model was confirmed by genomic PCR, sequencing, RT-PCR and proteomics. With this model, we found that the triglyceride (TG) levels are significantly lower in homozygote than that in wild type control. The results of lipoprotein analysis using ultracentrifugation and FPLC also show that TG-rich lipoproteins were lower in homozygote than that of wild type. TGs were not increased in homozygote even after feeding of food. Small LDL and HDL2 particles were increased in homozygote rabbits. In addition, increased apoE was shown in HDL3 particle from homozygote. These results suggest that apoA-II knock-in has critical roles in the metabolism of TG-rich lipoproteins. Roles of apoA-II only HDLs from homozygote on cholesterol efflux and atherogenesis are under investigation.
Abstract 193: Remnant-Like Particle Cholesterol, Low-Density Lipoprotein Triglyceride and Incident Cardiovascular Disease in the Atherosclerosis Risk in Communities Study: Can Genetic Variants Provide New Insights on Triglycerides and Atherogenic Lipoproteins?
Authors: Saeed Anum , Feofanova Eleana , Yu Bing , Virani Salim S , Sun Wensheng , Nambi Vijay , Heiss Gerardo , Guild Cameron S , Coresh Josef , Boerwinkle Eric , Ballantyne Christie M , Hoogeveen Ron
Published: July 2021

Recent genetic studies have focused on the atherogenicity of remnant-like particle cholesterol (RLPC) with paucity of data on TGs in LDL (LDL-TG). We examined the association of RLPC and LDL-TG with incident CVD and the genetic variants associated with their levels in the biracial ARIC study.

Fasting plasma RLPC and LDL-TG were measured by automated homogeneous assays (Denka Seiken, Tokyo) in 9334 men and women without prevalent CVD at baseline. Incident CHD and stroke over 16-y follow-up were obtained from medical records. Associations between LDL-TG and RLPC and the genomic variants (Illumina HumanExome Beadchip) were assessed using single variant analysis for common variants and gene-based burden tests for rare variants. Variants with minor allele frequency >1% were analyzed individually.

RLPC and LDL-TG were correlated with elevated TG (r=0.85 and 0.64, p<0.0001). In minimally adjusted analyses, both were associated with CVD risk, but after adjusting for traditional CVD risk factors including lipids, only LDL-TG was positively associated with incident CHD (HR 1.28 [95% CI 1.10-1.50]) and stroke (HR 1.47 [95% CI 1.13-1.92]; p<0.01). Single variant tests showed a common
APOE
variant (rs7412) had the strongest association with LDL-TG and RLPC in both races (p<5x10
-8
). Since rs7412 defines apoE isoforms, we assessed
APOE
haplotypes and found E2/2 was associated with reduced LDL-TG and increased RLPC (Fig).

Although elevated TGs are associated with increased RLPC and LDL-TG, only LDL-TG remained significant for CVD risk in multivariable-adjusted models. ApoE2 locus variants were associated with RLPC and LDL-TG, but individuals with apoE2/E2 had decreased LDL-TG and increased RLPC. These findings, and prior studies showing that apoE2 is associated with lower CVD risk, suggest that the increased risk of CVD with high TGs may be related more to LDL-TG than RLPC. Further research is needed to understand whether LDL-TG plays a causal role in CVD and is a target for therapy.
Abstract 194: Lipid and Apolipoprotein Size Profile Differences Between Low and High Body Mass Index Individuals in Response to Fat Challenge
Authors: Schieltz David , Kuklenyik Zsuzsanna , Parks Bryan A , Gardner Michael , Toth Christopher , Jones Jeffrey , Andrews Michael , Rees Jon , McWilliams Lisa , Barr John , Pirkle Jim L
Published: July 2021

Clearance of elevated triglycerides (TG) level in response to fat challenge is a clinically significant indicator of CVD risk. HDL can play a key role in facilitating TG metabolism explaining the reverse correlation of HDL levels with CVD risk. In this study 6 individuals, low BMI (21-25) and 3 high BMI (30-49) were challenged with a meal containing ~90 g of total fat. Blood samples were drawn in fasting state before meal and 3 times after meal in 2, 4 and 6 hours. Serum was analyzed by a quantitative, multiplexed analytical workflow which included analysis of whole serum and the separation of lipoproteins by asymmetric flow field-flow fractionation where fractions were collected with 1 nm increments of 7-15 nm (HDL), 20-30 nm (LDL) and >30 nm lipoprotein classes. Dynamic light scattering was used to determine hydrodynamic size in each size fraction. Whole serum and individual fractions were analyzed to determine concentrations of apolipoproteins (apo A-I, A-II, A-IV, B, C-I, C-II, C-III and E), and non-polar lipids (FC, CE and TG), using two parallel liquid chromatography tandem mass spectrometry methods developed in our laboratory. In whole serum, the total TG levels for low and high BMI donors, relative to pre-meal levels (57-137 mg/dL and 111-183 md/dL), peaked at between the 2 and 4 hours post meal (103-214 mg/dL and 210-309 mg/dL). LCAT were lower (20%) and CETP levels were higher (30%), in low vs high BMI groups, but did not show change in repose to the fat challenge. In both low and high BMI groups, the most significant change between pre-meal and post-meal after 2 hours occurred in HDL-ApoC-I, HDL-ApoC-II, and HDL-ApoC-III levels mainly in the HDL size range of 9-12 nm (medium HDL) and at 13-16 nm (large HDL), with concurrent TG increase observed in LDL and remnant fractions (Prob-
Abstract 196: Acute Phase Response Transcription Factor CREBH Mediates Metabolic Inflammation to Hepatic VLDL Overproduction and Hyperlipidemia
Authors: Song Yongyan , Zhao Miaoyun , Khound Rituraj , Su Qiaozhu
Published: July 2021

Hyperlipidemia is a major complication of insulin-resistant states, such as obesity and type 2 diabetes, and contributes to an increased risk of cardiovascular disease. One key mechanism underlying the hyperlipidemia in metabolic syndrome is the overproduction of apolipoprotein B (apoB), the essential structural protein of the triglyceride-rich lipoproteins, such as very-low-density lipoproteins (VLDL) and chylomicrons. Currently, the underlying mechanisms for the overproduction of VLDL in metabolic syndrome are not fully understood. In this study, we demonstrated that the cAMP responsive element binding protein H (CREBH), an acute phase transcription factor, enhanced VLDL assembly and secretion by upregulating apoB expression, which contributed to hyperlipidemia associated metabolic inflammation. Specifically, we showed that over-expression of CREBH significantly induced mRNA and protein expression of apoB in McA-7777 cells. A luciferase assay further revealed that the presence of CREBH positively enhanced the activity of the apoB gene promoter. Genetic depletion of CREBH in mice resulted in significant reduction in expression of hepatic apoB mRNA. Moreover, challenging mice with a fat load via oral gavage upregulated VLDL secretion in wild type but not in the CREBH-null mice, suggesting the essential role of CREBH in apoB expression. Inflammatory cytokine TNFα treatment activated hepatic CREBH expression in wild type mice which subsequently enhanced hepatic apoB biosynthesis and VLDL secretion. This phenotype was not observed in CREBH-null mice. Metabolic inflammation induced by LPS or HFD also resulted in overproduction of apoB and hyperlipoproteinemia in wild type but not in CREBH-null mice.

Conclusion:
This study demonstrated that, for the first time, CREBH could be a mediator between metabolic inflammation and hepatic VLDL overproduction in chronic metabolic disorders. This novel finding establishes CREBH as the first transcription factor that regulates apoB expression on the transcription level and the subsequent VLDL biosynthesis in response to metabolic inflammation, and further provides novel mechanistic insight into the pathogenesis of hyperlipidemia in metabolic syndrome.
Abstract 197: Characterization of the Effects of Angiopoietin-Like 3 on Plasma Lipids and Lipoproteins in Humans
Authors: Minnier Jessica , Fazio Sergio , Shapiro Michael , Tsimikas Sotirios , Arca Marcello , Tavori Hagai
Published: July 2021

Angiopoietin-like 3 (ANGPTL3) deficiency due to loss-of-function (LOF) gene mutations causes familial combined hypobetalipoproteinemia type 2 (FHBL2) in homozygotes and compound heterozygotes, with a lipid phenotype much milder in heterozygotes. We aim to determine whether a critical reduction in plasma ANGPTL3 levels is a major determinant of FHBL2 lipid phenotype. We studied 126 subjects from 19 families with ANGPTL3 LOF mutations. Individuals homozygous for mutations in ANGPTL3 manifest the full FHBL2 phenotype of reduced total cholesterol, triglycerides, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol (-C) and particle concentration (-p), and LDL size. Heterozygotes only displayed with low total cholesterol, HDL-C, HDL-p and VLDL-p compared with non-carriers. Using multivariate adaptive regression splines, we found that: (
i
) total cholesterol, triglycerides, LDL-C, HDL-C, HDL-p, and LDL size correlated with ANGPTL3 but only for levels below 25% of normal (<60 ng/dl); (
ii
) VLDL-p and LDL-p correlate with ANGPTL3 irrespective of its plasma levels; and (
iii
) homozygotes exhibited reduced levels of mature proprotein convertase subtilisin/kexin type 9 (PCSK9), a known regulator of plasma LDL-C levels. These results indicate that the full FHBL2 phenotype seen in homozygous carriers of LOF mutations in ANGPTL3 is caused by a critical reduction of more than 75% of its plasma levels, thus uncovering the relationship between mutation status, plasma ANGPTL3 concentrations, and the lipid phenotype. Furthermore, our study suggests that the low plasma LDL seen in homozygous carriers of ANGPTL3 LOF mutations is mediated by the modulation of plasma PCSK9.
Abstract 202: Synergistic Protective Effects of Statin and Angiotensin Receptor Blocker for the Progression of Atherosclerosis
Authors: LEE Seul-gee , Bong Sung-Kyung , Lee Oh-Hyun , Kim Choongki , Oh Jaewon , Park Sungha , Lee Sang-Hak , Kim Jung-Sun
Published: July 2021

Objective:
Both statin and angiotensin receptor blocker (ARB) are well known to prevent the progression of atherosclerosis. However, the additive effects of anti-atherosclerosis at early period are not fully investigated. Therefore, we tried to investigate whether an anti-atherosclerotic effect of statin and ARB could be synergistic and clarify its mechanisms.

Methods:
Atherosclerotic plaques were developed at 23 rabbits’ iliac arteries with a high cholesterol (HC) diet and balloon inflation. We applied with 5 different treatment for 4 weeks; group 1 [n=5, HC diet]; group 2 [n=3, a regular chow diet]; group 3 [n=5, HC diet and rosuvastatin (R, 10 mg/kg)]; and group 4 [n=5, HC diet and olmesartan (O, 20 mg/kg)]; group 5 [n=5, HC diet and R + O]. End of the period, optical coherence tomography (OCT) was performed to detect the plaque characteristics and all tissues were harvested for histological evaluation.

Results:
Histological analysis demonstrated that atherosclerotic plaque lesions (Intima/plaque ratio, %) were decreased in the combination group compared to the statin (p=0.001) or ARB group (p=0.17). The macrophage infiltration of RAM11 staining was not significantly different between combination and each treatment group (mean±SEM=31.8±4.8% (R+O) vs. 38.1±6.53% (R), p=0.35 or 35.1±2.9% (O), p=0.62). Relative area of macrophage M1 polarization (iNOS staining) was significantly lower in the combination group compared to ARB group (3.2±0.5% (R+O) vs. 4.6 ±0.4% (R), p=0.09 or 5.2±1.0% (O), p=0.03). M2 macrophage polarization (Arg-1 staining) revealed increase in the combination group compared to each treatment group (17.7±3.0% (R+O) vs. 7.9±0.7% (R), p=0.002 or 11.9±2.8% (O), p=0.05)

Conclusion:
The combination of statin and ARB appeared more beneficial effects to reduce plaque formation, which may be associated with modulation of macrophage subtype in early period.
Abstract 204: Prioritizing Genome-Wide Association Study Lipid Loci With Global Gene Networks
Authors: Nguyen Jennifer , Parks Brian W
Published: July 2021

Large genome-wide association studies (GWAS) conducted in humans by the Global Lipids Genetics Consortium (GLGC) have identified more than 150 genome-wide significant loci that are associated with variation in plasma lipid levels (cholesterol, LDL/HDL-cholesterol, and triglycerides). Some loci contain genes with well-described roles in lipid metabolism, such as
CETP
,
LDLR
, and
APOB
; however, for many genome-wide significant loci, there is no clear causal gene. To test the hypothesis that lipid genes in the liver are co-regulated, we constructed global co-expression gene networks from genome-wide gene expression data obtained from the livers of multiple independent mouse genetic crosses. All together, we constructed global gene networks from eight distinct studies, representing more than 800 unique mice of diverse genetic backgrounds. For all studies analyzed, we identified a module (or sub-network) of genes that is significantly enriched (p<1x10
-15
) functionally for cholesterol biosynthesis and metabolism genes. This module ranges in size from 70 to 824 genes across the eight studies and contains all genes involved in the cholesterol biosynthesis pathway (
Acat2, Hmgcs2, Hmgcr, Lss, Sc5d, etc.
). This module also contains many genes involved in the regulation of cholesterol metabolism, such as
Ldlr
,
Pcsk9
, and
Insig1
. Because of the significant enrichment of cholesterol genes in this module, we have begun to cross-reference all genes in the module against the GLGC lipid GWAS data. Through this analysis, we have identified genes of unknown function that are clearly located within genome-wide significant lipid loci as well as sub-threshold (suggestive significant) lipid loci. Among the genes we identified was
Sestrin1
, which was located within a clear sub-threshold locus associated with plasma cholesterol (rs12206606; p=1.4 x 10
-5
). In conclusion, our studies provide a framework to identify causal genes within reported lipid GWAS loci as well as to identify novel sub-threshold loci associated with variations in lipids among humans. We illustrate the approach by identifying
Sestrin1
within a sub-threshold locus associated with plasma cholesterol levels and show that
Sestrin1
is transcriptionally regulated in the liver by dietary cholesterol.
Abstract 206: Intestinal LXR Directs Absorbed Lipids to Storage
Authors: Schlegel Amnon
Published: July 2021

Post-prandial hypertriglyceridemia has emerged as a cardiovascular risk factor with limited therapeutic options. We have taken genetic and physiological approaches to identify and characertize novel factors that regulate intestinal lipid handling in the hope of revealing cellular pathways that dampen delivery of dietary lipids, opening the therapeutic window to blunting post-prandial lipid excursions. Liver X receptors (Lxrs) are master regulators of cholesterol catabolism, driving the elimination of cholesterol from the periphery to the lumen of the intestine. These nuclear hormone receptors are activated by oxysterol, ligands that accumulate when excess cholesterol is present. Loss of
nr1h3
function causes anticipated gene regulatory changes and cholesterol intolerance, collectively reflecting high evolutionary conservation of zebrafish Lxra function. Intestinal
nr1h3
activation delays transport of absorbed neutral lipids, with accumulation of neutral lipids in enterocyte cytoplasmic droplets, as visualized with histological stains, live fluorescent lipid probes, and ultrastructural analyses. This delay in transport of ingested neutral lipids protects animals over-expressing
nr1h3
in the intestine from hypercholesterolemia and hepatic steatosis induced by a high-fat diet. On a gene regulatory level, Lxra induces expression of
acsl3a
, which encodes acyl-CoA synthetase long-chain family member 3a, a lipid droplet-anchored protein that directs fatty acyl chains into lipids. We have identified a gene regulatory network in enterocytes whereby the pace of delivery of dietary lipids is gated by Lxrs. Development of pharmacological agents to activate Lxrs has been hindered by synthetic Lxr agonists’ induction of hepatic lipogenesis and hypertriglyceridemia. The development of intestine-limited Lxr agonists could be of therapeutic value in blunting post-prandial hypertriglyceridemia.
Abstract 207: ApoC-II Mimetic Peptide Blunts Post-Prandial Hypertriglyceridemia in Mice
Authors: Wolska Anna , Sakurai Toshihiro , Sviridov Denis , Pryor Milton , Lo Larry , Ghosh Soumitra , Devalaraja Madhav , Remaley Alan T
Published: July 2021

Post-prandial hypertriglyceridemia is as an important CVD risk factor. We recently described an apoC-II mimetic peptide (18A-CII) that activates LPL and lowers plasma TG in apoC-II-KO mice. To investigate 18A-CII as a therapy for post-prandial hyperTG, we investigated its effect on several mouse models after a fat challenge test. After an oral gavage with vegetable oil (10 μL/gram), plasma TG increased by 3 h in C57BL/6 mice by at least 5-fold but when mice were injected 30 min before gavage with 18A-CII (1 μmoL/kg; IP or SQ), the post-prandial TG rise was almost completely blunted. Similar results were found in apoE-KO mice, indicating that the effect of 18A-CII is independent of apoE. 18A-CII did not appear to work at the level of TG absorption, because it also rapidly accelerated plasma clearance of TG after IP injection of 20% Intralipid. Addition of exogenous LPL to plasma samples collected after IP injection with Intralipid revealed that lipoprotein particles from mice treated with 18A-CII were better substrates for LPL. The rise in plasma TG after IP injection of Intralipid was only partially blocked by about 50% when mice were co-injected with the LPL inhibitor Triton WR1339, indicating that 18A-CII also works by an LPL-independent mechanism. Incubation of human plasma with exogenous 18A-CII at doses comparable to that achieved
in vivo
in mice showed that 18A-CII binds to all lipoproteins, including HDL and causes the displacement of apoC-I and apoC-III. Furthermore, the
in vitro
inhibition of LPL from the addition of exogenous apoC-III could be overcome by adding 18A-CII at a molar ratio of at least 1:10 compared to apoC-III. In summary, the18A-CII mimetic peptide at relatively low doses can accelerate the clearance of post-prandial TG after a fat load. It does so in part by activating LPL but also by relieving the inhibition of LPL by apoC-III and possibly by also blocking the other known LPL-independent effects of apoC-III and possibly apoC-I in delaying the post-prandial clearance of TG.