bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 137: Comparing Blood Pressure Control in Diabetic Versis Nondiabetic Veterans Following Percutaneous Coronary Intervention
Authors: Challa Karthik , Ladani Amit , McGraw Sloane , Shivaraju Anupama , Shroff Adhir
Published: July 2021

Background:
As per the US Joint National Committee VII (JNC-7) recommendations, patients with known underlying coronary artery disease and diabetes should have goal blood pressures (BP) of systolic (SBP) <130 and diastolic (DBP) <80 to decrease morbidity and mortality associated with cardiovascular disease. In addition to lifestyle modification, these goals can be attained by use of multiple classes of drugs including beta-blockers (BB), angiotensin-converting-enzyme inhibitors/angiotensin receptor blockers (ACE-I/ARB), calcium channel blockers (CCB), diuretics and nitrates.

Methods:
We conducted a retrospective cohort study focusing on the attainment of the JNC-7 guidelines, comparing outcomes between 355 diabetic to 580 non-diabetic patients undergoing PCI between September 2004 and January 2011 at the Jesse Brown Veterans Affairs Hospital in Chicago, IL. BP measurements and antihypertensive medications pre and post PCI at 6-month follow-up were documented.

Results:
Among the diabetic population, the mean SBP decreased from 136 to 131 mmHg (p = 0.0007) and mean DBP decreased from 73 to 70 mmHg (p = 0.0005). In the non-diabetics, the mean SBP decreased from 133 to 127 mmHg (p < 0.0001) and the mean DBP decreased from 73 to 70 mmHg (p < 0.0001). With regards to JNC-7 guidelines, the percent of diabetics at SBP goal increased from 39% to 49% (p = 0.0053) and percent at DBP goal increased from 73% to 82% (p = 0.0098). In non-diabetics, percent at goal for SBP increased from 45% to 57% (p < 0.0001) and percent at DBP goal increased from 68% to 76% (p = 0.0009). Among diabetics, there was a statistically significant (p <0.0001) increase in use of BB from 79% to 92%. In non-diabetics, there was a statistically significant (p <0.0001) increase in use of BB from 66% to 87% and ACE-I/ARB from 51% to 70%.

Conclusions:
In both groups undergoing PCI, SBP and DBP improved with more patients achieving JNC-7 targets. Among diabetics, there was a significant increase in utilization of BB. Among non-diabetics, there was a significant increase in utilization of BB and ACE-I/ARB.
Abstract 156: Cholesterol-Induced IL-1 Expression Promotes Regulatory T Cells to Effector T Cells Redifferentiation in Atherosclerotic Lesions
Authors: Maganto-Garcia Elena , Tarrio Margarite L , Grabie Nir , Bu De-xiu , Lichtman Andrew H
Published: July 2021

Regulatory T cells (Treg) can suppress proatherogenic T cell responses, similar to their established function in regulating autoreactive T cells in immune-mediated/inflammatory disease. We have shown that hypercholesterolemia induces an accumulation of Treg in atherosclerotic aorta, but the Treg content is not maintained over time under sustained hypercholesterolemic conditions. Significantly, effector T cells (Teff) content in the aorta increased while Treg content went down concomitant with increase of the lesion size over this time period. Previous studies have shown that Treg change phenotype in vivo under inflammatory conditions and some studies indicate that IL-1 promotes differentiation of Th17 cells both from naïve precursors, and the conversion of Treg to Th17. The general hypothesis for this study is that the loss of lesional Treg over time may reflect a conversion of recruited Treg into Teff cells, due to the influence of the lesional inflammatory conditions, including production of IL-1. We have conducted preliminary studies in vitro showing that cholesterol crystals (CHC)-induced macrophage inflammasome activation modifies Treg phenotype. Thioglycollate elicited peritoneal macrophages from wildtype mice produced high levels of IL1 after CHC treatment compared to macrophages from caspase-1-null mice and this response induced IL-17 and IFNgamma expression by purified Treg from Foxp3-GFP TCR transgenic mice. We have also demonstrated, by confocal microscopy of sections from spleen or aortic arch of Ldlr
-/-
mice under cholesterol diet for 10 weeks, that macrophages but not dendritic cells uptake CHC in vivo. Moreover, IL-1 gene expression in aortas of Ldlr
-/-
mice is minimal after 4 weeks of cholesterol diet, but significantly rises after 8 weeks. This result correlated also with an increase of the levels of IFNgamma and Tbet and changes in numbers of IL17 or Foxp3 positive cells observed in collagen digest of aortas from Ldlr-/-/Foxp3-GFP/IL17-RFP. These results indicate that arterial IL-1 expression, which can be induced by CHC in macrophages under hypercholesterolemic conditions, accompanies a reduction in the Treg:Th1 ratio. Current work is focused on determining if atherosclerotic lesion development can be blocked by maintaining a sufficient ratio of atheroantigen-specific Treg:Teff cells through pharmacologic manipulation of inflammasome activation.
Abstract 17: Reverse Cholesterol Transport Relies on a Functional Lymphatic Network
Authors: Martel Catherine , Platt Andrew M , Westerterp Marit , Bittman Robert , Tall Allan R , Chen Shu-Hsia , Sorci-Thomas Mary , Swartz Melody A , Randolph Gwendalyn J
Published: July 2021

Introduction:
HDL accepts free cholesterol from cells within extravascular tissues and transports it to the liver for excretion. Little is known about how HDL-C leaves such tissues to reach the blood. HDL-C is found in lymph, but it is unclear if lymphatics are critical for mobilization of HDL-C from tissues.

Methods & Results:
We employed models of disrupted lymphatic drainage--one a surgical resection of lymphatics in the mouse tail and the other a genetic lack of skin lymphatics due to a mutation in VEGFR3--to quantify the role of lymphatic vessels in movement of HDL-C out of skin to plasma. We implanted bone marrow macrophages loaded with [
3
H]-cholesterol in skin sites and then quantified [
3
H]-cholesterol in plasma, liver, and feces. [
3
H]-cholesterol movement into plasma was reduced by 80% in both models of disrupted lymphatics, and this result was unaffected by conditions of a leaky blood vessels. To ensure that this reduction reflected events between efflux and appearance of cholesterol in plasma rather than altered macrophage cholesterol efflux, we substituted [
3
H]-cholesterol-loaded macrophages with congenic macrophages loaded with a fluorescently modified cholesterol compound that allowed us to monitor ABCA1/ABCG1-dependent efflux from individual macrophages in the same assays and models. Flow cytometric analysis of bodipy-cholesterol-loaded macrophages retrieved from injection sites revealed similar efflux in mice lacking functional lymphatics as in controls.

Conclusions:
Mobilization of HDL-C from extravascular tissues to plasma occurs through the lymphatic vasculature. Recent studies show that transport through lymphatic vessels is compromised by hypercholesterolemia. Hypercholesterolemia-induced impairments in lymphatic function may retard clearance of cholesterol from extravascular tissues, possibly including atherosclerotic plaques.
Abstract 18: Apo(a) Isoform Size Is Associated with Myocardial Infarction in a Study of an Admixed Population from the Central Valley of Costa Rica
Authors: Bare Lance A , Rowland Charles M , Koschinsky Marlys L , Boffa Michael B , Tong Carmen H , Arellano Andre R , Catanese Joseph J , Shiffman Dov , Devlin James J , Campos Hannia
Published: July 2021

Objective:
Lipoprotein(a) [Lp(a)] particles contain a single apolipoprotein(a) [apo(a)] molecule. Both plasma Lp(a) levels and apo(a) isoform size are associated with myocardial infarction (MI). We asked whether Lp(a) level and apo(a) isoform size are independently associated with MI in an admixed population (African, Amer-indian, Southern European) from the Central Valley of Costa Rica.

Methods:
Apo(a) isoform size is determined by the number of kringle IV type 2 (KIV-2) repeats in the protein, and when two different alleles are expressed, the smaller isoform is typically expressed at higher levels. We assessed isoform size by Western blot and Lp(a) levels by immunoturbidometric assay in randomly selected MI cases and population-based controls from a study of 4186 Central Valley Costa Ricans. Association with MI was assessed using logistic regression models that included Lp(a) level and the size of the smaller isoform as explanatory variables. A model including adjustment for local ethnicity determined by principal component analysis of 41 SNPs within 410 kilobases of the gene encoding apo(a) [LPA] was performed on a subset of subjects with genotypes for all 41 SNPs.

Results:
For 433 subjects, for whom one or both apo(a) isoform sizes were determined by Western blot, we found that both size of the smaller expressed apo(a) isoform and Lp(a) level were associated with MI: OR=1.48 (p= 0.021) per decrease of 5 KIV-2 repeats and OR=1.27 (p=2.8x10-5) per 10 mg/dL increase in Lp(a). In multivariable analysis, the size of the smaller apo(a) isoform was associated with MI after adjustment for Lp(a) levels (OR=1.29; p= 0.034). Similarly, Lp(a) levels remained associated with MI after adjustment for the size of the smaller apo(a) isoform (OR=1.19; p=0.005). Further adjustment for local ethnicity did not appreciably change the association of the smaller apo(a) isoform with MI (OR= 1.38;p= 0.056) or the association of Lp(a) levels with MI (OR=1.11, p=0.18). Apo(a) isoform size was strongly associated with Lp(a) levels (9.6 mg/dL increase in Lp(a) per decrease of 5 KIV-2 repeats (p=1.7x10-24).

Conclusion:
Apo(a) isoform size is associated with MI after adjustment for Lp(a) in a population from the Central Valley of Costa Rica. The hypothesis that apo(a) isoform size is associated with MI independent of Lp(a) levels appears worthy of further study.
Abstract 201: Adventitial Nab-Rapamycin Injection Reduces Porcine Femoral Artery Luminal Stenosis Induced by Balloon Angioplasty via Inhibition of Medial Proliferation and Adventitial Inflammation
Authors: Gasper Warren J , Jimenez Cynthia A , Seward Kirk , Conte Michael S , Owens Christopher D
Published: July 2021

Introduction:
Balloon angioplasty of peripheral arteries induces a substantial inflammatory response and has a high rate of restenosis. Our hypothesis was that peri-adventitial injection of the antiproliferative, immunosuppressant drug rapamycin would reduce luminal stenosis in a porcine femoral artery model of balloon angioplasty injury.

Methods:
16 male crossbred swine had a balloon angioplasty overstretch injury of the femoral artery followed by peri-adventitial injection of saline or 500μg of nanoparticle albumin-bound (nab) rapamycin with an intraluminal microinfusion catheter. Qualitative and quantitative assessments of the femoral arteries, including histomorphometric measurements, were performed on H&E, Masson’s trichrome or immunohistochemistry stained sections. Blood and tissue rapamycin concentration were assessed by liquid chromatography and mass spectrometry at 1h, 3d, 8d and 28d.

Results:
There was 100% procedural success with periadventitial injection. At 28 days, femoral arteries treated with nab-rapamycin had a 42% reduction in luminal stenosis, 19.5 ± 3.3% vs 11.4 ± 0.84%, p=0.01 t-test (figure). Nab-rapamycin also significantly reduced medial fibrosis by qualitative analysis, p<0.0001 t-test, and media cell proliferation by Ki-67 staining, p=0.02 t-test. There were significantly fewer adventitial leukocytes at 3 days, p=0.03 t-test, but no difference at 28 days. One hour after injection, the perivascular concentration of nab-rapamycin was 1500-times higher than serum and rapamycin persisted in the tissue for at least 8 days. Rapamycin was not detectable in serum or tissue at 28 days. There was no difference in endothelial coverage or regeneration by Factor VII and H&E staining at 3, 8 or 28 days.

Conclusions:
A single periadventitial dose of nab-rapamycin significantly reduced luminal stenosis in a porcine model of femoral artery balloon angioplasty injury. The observed reduction in early adventitial infiltration by leukocytes and decrease in medial cell proliferation and fibrosis at 28 days suggests a mechanism by which nab-rapamycin may have an effect. Adventitial injection with an intraluminal microinfusion catheter is safe and efficient and represents an alternative to stent- or balloon-based local drug delivery.
Abstract 222: D-Series Resolvins Modulate Postinjury Signaling Pathways in Vascular Smooth Muscle Cells
Authors: Miyahara Takuya , Chatterjee Anuran , Chen Mian , Runge Sara , Conte Michael S
Published: July 2021

Introduction:
Recent work suggests that the resolution of inflammation is an active process involving specialized molecules that are locally generated. D-series resolvins (RvD) are endogenous pro-resolving lipid mediators derived from the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). VSMC activation following vascular injury leads to neointimal hyperplasia which may reflect a relative “resolution deficit” in the vessel wall.

Objectives:
We sought to determine the effects of RvD on injury-related inflammatory and growth factor signaling pathways in VSMC, using
in vitro
and
in vivo
models.

Hypothesis:
We hypothesize that RvD will attenuate VSMC activation responses and modulate vascular injury.

Methods:
Human VSMC were treated with cytokines (TNF-α, IL-1β), growth factors (PDGF, AngII) or oxidant stress (H
2
O
2
) in the presence of RvD1 or RvD2 (.01-100 nM), and phenotype was assessed by cytotoxicity, proliferation, migration (transwell), monocyte adhesion (U937) and adhesion molecule expression (qPCR and flow cytometry) assays. NZW rabbits (N=4) underwent bilateral femoral balloon injury with intraluminal delivery of RvD2 (10 nM vs vehicle control) to examine acute effects (3 days) on the injury response.

Results:
RvD treatment of VSMC produced dose-dependent inhibition of VSMC proliferation (max %inhibition: RvD1 58.8%, p<0.01, RvD2 63.1%, p<0.01), migration (RvD1 66.1%, p<0.01%, RvD2 68.8%, p<0.01), monocyte adhesion (RvD1 36.0%, p<0.01, RvD2 41.0%, p<0.01), and inducible adhesion molecule expression (VCAM-1: RvD1 70.5%, p<0.01, RvD2 77.2%, p<0.01; ICAM-1: RvD1 65.4%, p<0.01, RvD2 72.1%, p<0.01) without measurable cytotoxicity. In the rabbit model of balloon arterial injury, proliferation was significantly reduced at 3 days in RvD2 treated vessels (%inhibition 57%, p<0.01).

Conclusion:
RvD broadly inhibit VSMC activation responses and may modulate the vascular injury response
in-vivo
.
Abstract 228: Secretion of Bone Marrow--Derived Cells Contributes to Pressure-Overload--Induced Left Ventricular Hypertrophy
Authors: Yang Fanmuyi , Dong Anping , Smyth Susan
Published: July 2021

Bone marrow derived cells, especially inflammatory cells, may contribute to the pathological progression of pressure overload induced left ventricular (LV) hypertrophy and cardiac fibrosis. Previously, we reported that inflammatory cell accumulation and upregulation of cytokines in hearts of mice that had undergone transverse aortic constriction (TAC) surgery predicted later cardiac hypertrophic and fibrotic remodeling. However, the underlining mechanisms are still not clear. One potential mechanism for inflammatory cells to modulate their environment and affect surrounding cells is through release of cargo stored in granules. Jinx mice - which contain a single point mutation in Unc13d encoding the Munc13-4 protein, a limiting factor in vesicular priming and fusion - have defects in granular secretion in hematopoietic cells, such as platelets, NK cells, and neutrophils. In the current study, we investigated the role of bone marrow cell granule secretion in TAC-induced LV remodeling by creating with bone marrow transplantation chimeric mice specifically lacking Munc13-4 in marrow derived cells. Both wild-type mice (WT) that were transplanted with WT bone marrow and WT mice that received Jinx bone marrow developed LVH and a classic fetal reprogramming response early (7 days) after TAC. However, at later times (5 weeks), mice lacking Munc13-4 in bone marrow-derived cells failed to sustain the cardiac hypertrophy observed in mice with WT bone marrow. No difference in cardiac fibrosis was observed at early or late times. These results suggest that sustained LVH in the setting of pressure overload depends on factor(s) secreted from bone marrow-derived cells, likely from either platelets, NK cells and/or neutrophils. Inhibiting granule cargo release may represent a novel therapeutic target to prevent the development of LVH.
Abstract 231: Allogeneic Mesenchymal Stromal Cells Derived from the Chorionic Villi of Human Placentae Are a Superior Source of Progenitor Cells for Angiogenic Therapies
Authors: Jester Andrea L , Saadatzadeh M Reza , Johnstone Brian , Njoku Victor , Bhavsar Janak K , Ofstein Richard H , Tempel Zachary S , Traktuev Dmitry O , Yoder Mervin C , March Keith L , Murphy Michael P
Published: July 2021

Introduction:
Autologous bone marrow mesenchymal stromal cells (ABMSCs) have been the focus for the treatment of myocardial and limb ischemia. The utility of ABMSCs is limited because, in addition to requiring an invasive procedure to harvest, the number and the regenerative capacity of ABMSCs decrease with age and comorbid conditions. A redirected focus on alternative sources of MSCs is warranted. Here we compare MSCs from adipose tissue (ASCs) and human placenta (pMSCs) for angiogenic therapies.

Methods:
ASCs and pMSCs were characterized using culture assays, flow cytometry and immunofluorescent staining. Immunodulatory properties were assessed using mixed lymphocyte reactions (MLR). Inflammatory and angiogenic cytokines were quantified using ELISA. The ability of ASCs and pMSCs to restore perfusion in a murine model of hindlimb ischemia was compared.

Results:
ASCs and pMSCs expressed cell surface antigens consistent with MSCs. In permissive cultures, ASCs and pMSCs stained positive for Oil Red O (adipogenic), Alcian Blue (chondrogenic) and alkaline phosphatase (osteogenic) in addition to neural and myogenic lineages. Immunofluorescent staining demonstrated that PMSCs but not ASCs express embryonic stem cell antigens Oct-3/4, Nanog, SSEA-3 and SSEA-4. ASCs and pMSCs demonstrated immunosuppressive properties in MLRs (n=3). There was a significant reduction of MNC proliferation when cultured with pMSCs (96.5 ± 3.7 % reduction;
p
< 0.001) or ASCs (93.27 ± 2.3 % reduction;
p
< 0.001). When compared to ASCs, pMSCs (n=3) secrete significantly greater quantities of VEGF (4.4 ± 0.13 vs. 2.5 ± 0.031 ng/mL;
p
< 0.001), HGF (2.4 ± 0.114 vs. 1.6 ± 0.173 ng/mL;
p
< 0.001), and Ang-1 (4.6 ± .23 vs. 1.6 ± 0.016 ng/mL;
p
< 0.001) in hypoxia. In NOD/SCID mice with femoral artery ligation (n=6), those treated with pMSCs demonstrated a significant improvement in perfusion compared to those treated with ASCs at 35 days post-treatment (56.18 vs. 40.82;
p
< 0.001).

Conclusion:
In conclusion, while both ASCs and pMSCs have potent immunosuppressive properties, pMSCs are superior in the production of angiogenic cytokines and restoration of perfusion in ischemic tissue. PMSCs can be banked and thus made readily available for the treatment of acute ischemic syndromes.
Abstract 256: 2D Ultrasound Measurements Can Quantify Relative Single-Plane Circumferential Strain in an Abdominal Aortic Aneurysm Model
Authors: Mix Doran S , Schwarz Karl , Gillespie David , Chandra Ankur
Published: July 2021

OBJECTIVES:
Current size-based assessments of AAA rupture potential do not accurately identify all patients at risk. True AAA rupture potential is related to hemodynamic and geometric factors involving wall strain and compliance. Our hypothesis is that transcutaneous ultrasound-derived strain measurements can identify heterogeneous aortic wall compliance toward predicting future rupture.

METHODS:
A latex phantom with changes in wall thickness (0.05-0.25 inches) to simulate AAA morphology was tested on an
in vitro
hemodynamic simulator. A GE Vivid i ultrasound machine interrogated the phantom under uniform physiologic, pulsatile conditions. The circumferential strain and radial strain of uniform wall phantom versus asymmetric wall phantom was quantified.

RESULTS:
Maximum circumferential strain (MCS) of the uniform wall thickness phantom was evenly distributed at 3.5% with an AP wall strain difference of 2.3% (Figure 1A). Maximum radial strain (MRS) was evenly distributed at 9% with an AP wall strain difference of 0%. MCS and MRS of the asymmetric wall thickness phantom were significantly increased to 30% and 36% respectively at the thinned anterior wall. AP wall strain difference was 22% (Figure 1B) and 10% respectively.

CONCLUSIONS:
Using transcutaneous 2D ultrasound, we were able to quantify changes in strain due to wall compliance in an AAA phantom. Further development of this technology may provide for a non-invasive method of characterizing the hemodynamic and geometric properties of an AAA to predict rupture potential.
Abstract 260: Knockout of Apolipoprotein A-II Has Dramatic Effects on High-Density Lipoprotein Subspecies Distribution
Authors: Gordon Scott M , Reardon Catherine A , Getz Godfrey S , Davidson W S
Published: July 2021

High density lipoproteins (HDL) are a highly heterogeneous population of particles composed of various lipids and proteins. They have been demonstrated to possess a diverse variety of functional properties which are thought to contribute to protection against cardiovascular disease (CVD). Proteomics studies have identified up to 75 different proteins which can associate with HDL. The basis for the compositional diversity of HDL is not known but a better understanding will yield important information about its broad functional diversity. To investigate the impact of common HDL apolipoproteins on the distribution of other apolipoproteins, we have begun to systematically fractionate plasma from various HDL apolipoprotein KO mice. Plasma from apoA-I, apoA-IV and apoA-II global KO mice was applied to gel filtration chromatography to distinguish HDL size populations. HDL particles sequestered by a phospholipid binding resin were proteomically analyzed by electrospray ionization mass spectrometry. By comparing elution volume shifts (i.e. particle size variations) for each HDL protein between WT controls and the KO models, we assessed the impact of the deleted protein on HDL size distributions. Ablation of apoA-I, while decreasing total HDL phospholipid by 70%, had a surprisingly small impact on the distribution of the majority of other HDL associated proteins - affecting only 9 of them. Genetic apoA-IV ablation had a similar modest effect shifting a distinct subset of 9 proteins. However, loss of apoA-II, in addition to causing a similar 70% reduction in overall HDL phospholipids, affected the size distribution of some 45 HDL proteins (including several complement proteins and paraoxonase-1). These data suggest that apoA-I, while associated with the majority of HDL phospholipid, may actually interact with relatively few of the lower abundance proteins known to be associated with HDL. ApoA-II on the other hand, may interact with many of these, perhaps acting as a docking site or adaptor molecule.
Abstract 264: Differential Profiles of Thrombin Inhibitors (Dabigatran, Hirudin, Bivalirudin and Heparin) in the Thrombin Generation Assay (TGA) and Thromboelastography (TEG) in Vitro
Authors: Xu Yiming , Wu Weizhen , Plump Andrew S , Chintala Madhu , Ogletree Martin L , Chen Zhu
Published: July 2021

Thrombin is a central enzyme in haemostasis and thrombosis, and a proven target for anticoagulant therapies. Different classes of thrombin inhibitors, while exerting therapeutic benefits in most clinical trials, have different indications, dosing regimens, and bleeding complications. To gain more insight into the underlying mechanisms for their differential clinical profiles, we compared four marketed and representative agents, including dabigatran, hirudin, bivalirudin (direct thrombin inhibitors, DTIs), and heparin (an indirect thrombin inhibitor), in two in vitro spike-in assays with concentration titrations covering their therapeutic ranges. The two assays were the Thrombinoscope TGA with plasma, triggered by low tissue factor (1 nM TF), and TEG with whole blood, triggered by 1:8000 Recombiplastin (equivalent to low TF), with or without a threshold level of tPA to induce fibrinolysis. In TGA, the largest effect was prolongation of lag time, with the potency of the three DTIs rank-ordered as hirudin>dabigatran>bivalirudin; regarding peak, slope, and ETP, while complete inhibition was achieved with 1-2 μM dabigatran or hirudin, bivalirudin had no effect even at 4 μM, possibly due to its short half life in plasma. In TEG, the three DTIs prolonged clotting time (R) in the same rank order as TGA; for clot strength (MA), while all four agents reduced MA in synergy with tPA, only hirudin reduced MA without tPA, likely due to its highest potency. With tPA-induced fibrinolytic activity (Ly30), dabigatran and bivalirudin enhanced Ly30 (dabigatran>bivalirudin), but hirudin and heparin did not. This contrast might involve differential access to clot-bound thrombin. Heparin had a steep dose-response curve for both lag time in TGA and R in TEG, which is in line with its very narrow therapeutic index. All three DTIs, but not heparin, displayed the previously reported paradoxical increase in peak and slope in TGA in the low concentration range, suggesting this is indeed a class effect of DTI. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. These results in combination with known clinical properties are informative on efforts to define the optimal profiles of new anticoagulants.
Abstract 28: Serial in Vivo Imaging of Thrombus Inflammation Predicts Venous Thrombus Resolution
Authors: Kessinger Chase W , McCarthy Jason R , Lin Charles P , Henke Peter K , Jaffer Farouc A
Published: July 2021

Introduction:
Inflammation following deep vein thrombosis (DVT) critically modulates thrombus resolution. However it is unknown whether the degree of inflammation
in vivo
predicts the magnitude of thrombus resolution. Here, we utilize serial multichannel intravital fluorescence microscopy (IVFM) to spatially map and quantify macrophage infiltration in thrombus, and then to determine the degree of thrombus resolution
in vivo
.

Methods:
C57Bl/6 mice (n=5) underwent topical ferric chloride injury to induce femoral venous thrombosis. On day 3, mice were i.v. injected with macrophage-targeted nanoparticles (CLIO-AF555, ex/em 555/565nm). On day 4, survival IVFM of thrombus macrophages was performed. In addition, venography was concomitantly performed (pre-injection of FITC-dextran 45 minutes before IVFM, MW 2000 kD, 490/520nm) to quantify DVT area and length in vivo at day 4 and 6. IVFM image analysis was performed using ImageJ. Mid-luminal z-stacks (40 μm thickness) were analyzed. Thrombus ROIs were outlined using FITC-dextran generated angiograms, and used calculating macrophage target-to-background ratios (TBR) and thrombus area and length. Contralateral sham femoral veins were imaged as controls. Imaging was followed by histopathology and fluorescence microscopy of the cryosectioned tissue.

Results:
At day 4, the baseline femoral DVT area and length were 0.22±0.08mm
2
and 1.29±0.16mm (mean±sd). The day 4 macrophage activity was noted to be heterogeneous with peripheral infiltration of the thrombus, and occasionally with some deeper penetration into the thrombus interior. At day 6, the thrombus area and length were reduced compared to day 4 (p<0.005 for both). The day 4 thrombus macrophage TBR significantly predicted the degree of thrombus reduction (correlation of initial macrophage TBR and Δthrombus area, r=0.97, p=0.001).

Conclusion:
This serial in vivo assessment of murine DVT resolution demonstrates that degree of macrophage infiltration at day 4 predicts the magnitude of thrombus resolution at day 6. Molecular imaging of DVT inflammation
in vivo
could provide new insights into DVT resolution and the development of the post-thrombotic syndrome, and also in evaluating inflammatory-modulating therapies for improved DVT resolution.
Abstract 282: Murine Abcb1 Functions as an Efflux Transporter for Bile Salts After Chronic Administration of a Western Diet
Authors: Lee Stephen D , Thornton Sheila J , Wasan Kishor M
Published: July 2021

Rationale:
Removal of bile salts from the liver is the final step of the reverse cholesterol transport pathway. We studied the contribution of Abcb1 (P-glycoprotein), in bile acid efflux. Although a number of endogenous substrates have been postulated for Abcb1 based on
in vitro
evidence, studies using animal models have not supported these claims. Recent studies in mice demonstrated that in the absence of the Bile Salt Efflux Pump (Bsep), Abcb1 is required for removal of bile salts, especially when challenged with a cholic acid containing diet. To date, no study using atherogenic diets has demonstrated the role of Abcb1 in the removal of bile salts in the presence of functional Bsep.

Methods:
We fed male mice lacking both isoforms of Abcb1 (Abcb1a
-/-
/1b
-/-
) and wild-type controls a diet providing either 25% or 45% of the kcal from fat, supplemented with either normal chow or high levels of cholesterol (0.02% w/w or 0.2% w/w respectively) for nine weeks; n=5 per group. On the tenth week, we assessed the efflux of cholesterol, phospholipid and bile acids to the gallbladder. Enzymatic assays were used to measure cholesterol and phospholipid, the pool of bile acids was quantified by HPLC to determine the concentrations of the six most prevalent murine bile acids.

Results:
Abcb1 knockout mice have a >30% reduction in the moles of bile salt normalized to phospholipid relative to wild type mice after administration of diets containing either elevated fat or cholesterol (p<0.05). Neither the efflux of phospholipid, nor the molar composition of the six bile acids was affected by deletion of Abcb1.

Conclusions:
We conclude that Abcb1 is a secondary efflux mechanism required for the removal of bile acids after consumption of diets rich in fat and/or cholesterol. Although Abcb1 knockout mice have reduced total bile acids in the gallbladder, the molar ratio of the specific bile acids is the same as in the wild type mice. These data suggest that Abcb1 effluxes the six bile acids in a non-specific manner, unlike Bsep which preferentially effluxes hydrophobic bile acids. The lack of specificity demonstrated by Abcb1 is desirable for a low- affinity secondary efflux mechanism, which supplements Bsep activity in bile acid output.

Acknowledgments:
Canadian Institutes of Health Research, Michael Smith Foundation for Health Research
Abstract 3: Site-Specific Chlorination of ApoA-I, the Major HDL Protein, Is Associated with Impaired Sterol Efflux and Is Markedly Increased in Subjects with Coronary Artery Disease
Authors: Shao Baohai , Tang Chongren , Mayer Philip , Zhao Xue-Qiao , Heinecke Jay W
Published: July 2021

3-Chlorotyrosine is a specific product of myeloperoxidase (MPO), a heme protein expressed by macrophages in human atherosclerotic lesions. We have previously shown in vitro that chlorination of Tyr192 (3-chloroTyr192) together with methionine oxidation of lipid-free apoA-I impairs the apolipoprotein’s ability to promote cholesterol efflux from macrophages by the ABCA1 pathway. Using a sensitive and quantitative LC-MS/MS approach with selected reaction monitoring (SRM), we have recently demonstrated that Tyr192 is the major site of chlorination in apoA-I of HDL isolated from human atherosclerotic lesions. In a prospective study, we determined if: i) MPO contributes to the chlorination of apoA-I in circulating HDL; and ii) 3-chloroTyr192 serves as a marker for clinically significant cardiovascular disease. We quantified HDL oxidation and sterol efflux in three groups: i) stable CAD subjects, ii) acute coronary syndrome (ACS) subjects, and iii) healthy control subjects (N=20 per group). SRM analysis demonstrated that levels of 3-chloroTyr192 were markedly elevated in HDL isolated from subjects with CAD or ACS (p=0.009 and p=0.01 vs. control subjects, respectively). A key cardioprotective effect of HDL is its ability to promote cholesterol efflux from macrophages by the ABCA1 pathway. We found that the ABCA1-dependent sterol efflux capacity of HDL isolated from CAD and ACS subjects was significantly impaired. Importantly, sterol efflux capacity associated inversely with levels of 3-chloroTyr192 (R=-0.30, p=0.02), suggesting that oxidation of HDL by MPO contributes to the impaired cholesterol efflux capacity of HDL. Receiver operating characteristic curve analysis revealed that levels of 3-chloroTyr192 distinguished between CAD or ACS subjects and control subjects (AUC 0.76, p=0.0008 and AUC 0.73, p=0.004, respectively). These observations strongly suggest that MPO contributes to the generation of dysfunctional HDL in subjects with clinically significant atherosclerosis. They further indicate that quantification of 3-chloroTyr192 levels in HDL may provide a new approach to the diagnosis and treatment of CAD and ACS.
Abstract 300: Leptin Promotes the Osteoblast Differentiation and Mineralization of Primary Cultures of Vascular Smooth Muscle Cells by Regulating GSK-3β
Authors: Zeadin Melec G , Butcher Martin K , Werstuck Geoff H
Published: July 2021

Obesity is an independent risk factor for cardiovascular disease (CVD) although the precise molecular mechanisms that link obesity to CVD are not understood. Recent studies suggest that factors secreted by adipose tissue may play an adverse role in cardiovascular health. We have previously demonstrated that the adipocytokine, leptin, promotes vascular calcification in apolipoprotein E - deficient mice and that this increase in calcification is associated with an increase in the expression of several osteoblast-specific markers within the vessel wall. In an effort to understand the mechanism by which leptin exerts these effects, we cultured primary bovine aortic smooth muscle cells (BASMCs) in the presence of 0 to 2 μg/ml leptin for up to 12 days. Osteogenic differentiation of BASMCs was determined by an increase in the expression of osteoblast-specific markers, and the induction of both alkaline phosphatase activity and mineralization. Consistent with previous studies, we found that treatment of BASMCs with leptin induced osteoblast differentiation in a dose-dependent manner. To investigate the underlying mechanism, we examined changes in the expression levels of key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway. We found that exposure to leptin induced the Erk 1/2-dependent inactivation of GSK-3β, through Ser9 phosphorylation, and a subsequent increase in the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a > 2-fold increase in GSK-3β activity, a decrease in the expression of the osteoblast-specific marker, osteopontin, and a significant decrease in leptin-induced alkaline phosphatase activity. Together, our results provide a possible mechanism by which elevated leptin concentrations, associated with obesity, promote osteoblast differentiation, and vascular calcification in vivo.
Abstract 313: The Nuclear Liver X Receptor as a Potential Regulator of Interleukin 18 Binding Protein in the Lung
Authors: Kratzer Adelheid , Salys Jonas , Mouman Zakaria , Zamora Martin , Taraseviciene-Stewart Laima
Published: July 2021

Background and Objectives:
Second hand smoke (SHS) is a major risk factor for development of chronic obstructive pulmonary disease (COPD), including emphysema and chronic bronchitis in non-smokers. The role of liver X receptor (LXR) in the pathogenesis of COPD/emphysema is not well understood. Both LXR and LXR were found in the lung.

The alpha isoform is involved in the regulation of lipid metabolism whereas the beta isoform is involved in the regulation of inflammation. Here we investigate the effects of cigarette smoke (CS) on LXR activation as well as target gene and protein regulation
in vivo
and
in vitro
.

Methods:
Sprague Dawley rats were exposed to SHS for 2 months. Lung tissue and bronchoalveolar lavage fluid (BALf) cells were examined for LXR gene expression and emphysema development (measured by mean linear intercept). Rat alveolar macrophages (AM) and rat pulmonary microvascular endothelial cells (RPMVEC) were treated with cigarette smoke extract (CSE), LPS or DMHCA, a steroidal LXR agonist)
in vitro
and changes in LXR and its target gene and protein expression were examined.

Results:
LXR gene expression was impaired and the expression of the LXR target gene, ATP binding cassette transporter A1 (ABCA1), was significantly downregulated in BALf cells from SHS-exposed rat lungs and in rat AM treated with CSE. Real-time PCR revealed significant downregulation of LXR and upregulation of LXR in BALf cells after 1 month of SHS exposure. Similar changes were also observed in CSE-treated RPMVEC. There was a significant increase in the pro-inflammatory interleukin 18 (IL18) levels in BALf cells of SHS-exposed rats, whereas the levels of IL18 binding protein (IL18bp), an endogenous inhibitor of IL18, remained unchanged. Interestingly, activation of LXR led to upregulation of IL18bp in AM and peritoneal macrophages where potential LXR response element (LXRE) sites were indentified,
in silico
, in the promoter region of IL18bp. Additionally, emphysematous changes and foamy macrophage accumulation were observed in lungs of LXR-deficient mice.

Conclusions:
These findings implicate IL18bp as a new LXR target gene involved in the anti-inflammatory signaling pathway. Our data suggest that LXR agonist treatment may be beneficial in the prevention and treatment of CS-induced emphysema.
Abstract 314: D-4F Decreases Myocardial Inflammation in a Model of Murine Scleroderma by Decreasing Interferon Regulating Factor-5 Levels
Authors: Krolikowski John G , Jones Deron W , Pritchard Kirkwood A , Weihrauch Dorothee
Published: July 2021

D-4F, an ApoAI mimetic, has potent anti-inflammatory properties. It is reported to alter intracellular signaling cascades such as the Jak/Stat pathway which many consider to play an important role in autoimmunity. Interferon regulating factor 5 (IRF5) is a transcription factor that has recently been shown to serve as a critical switch in controlling macrophage phenotype. Interestingly, IRF5 is downstream of Jak/Stat. Previously, we showed that D-4F decreases inflammation and fibrosis in the myocardium of tight skin mice (Tsk
-/+
), an established murine model of scleroderma. As D-4F alters Jak/Stat signaling we hypothesize D-4F may inhibit IRF5 to decrease inflammation and fibrosis in the hearts of Tsk
-/+
mice. To test this hypothesis we performed 1) western blot analysis for IRF5 on myocardium of C57Bl/6J mice, Tsk
-/+
mice, and Tsk
-/+
mice treated with D4F; 2) immunoblots (IBs) for phosphoserine and ubiquitin on immunopreciptiates (IPs) for IRF5; 3) immunohistochemistry on myocardial sections for IRF5; and, determined D-4F interactions with IRF5 by biolayer interferometry (BLI). Our studies reveal that IRF5 levels in Tsk
-/+
myocardium were virtually the same as in the myocardium of C57Bl/6J mice and that D-4F treatments reduced IRF5 protein levels in the hearts of Tsk
-/+
mice by 70 % (p<0.002, n=3). IPs/IBs studies showed that IRF5 phosphoserine levels, an index of activation, in Tsk
-/+
mice were essentially the same as in C57BL/6J mice. D-4F treatments however, markedly reduced phosphoserine levels on IRF5 in Tsk
-/+
myocardium (∼50%, p<0.002, n=3). In addition, IRF5 ubiquination, which is required for transport into the nucleus, was increased in Tsk
-/+
mice by ∼3-fold with no signs of degradation, compared to the levels in C57BL/6J mice. In contrast, D-4F decreased IRF5 ubiquitination in Tsk
-/+
mice by ∼50% with signs of degradation. Interestingly, BLI studies suggested that D-4F binds to IRF5 irreversibly. Taken together these data suggest D-4F decreases myocardial inflammation and fibrosis by decreasing total IRF5 protein and IRF5 activation.
Abstract 320: Cardiovascular Phenotype of PCSK9 Knockout Mice
Authors: Papa Umberto , Toumaniantz Gilles , Loirand Gervaise , Costet Philippe
Published: July 2021

Proprotein convertase subtisilin kexin type 9 (PCSK9) promotes LDL receptor (LDLR) degradation and its absence confers life-long low LDL-cholesterol, protection against coronary artery disease. Afro-americans patients with nonsense PCSK9 mutations are less prone to hypertension and plasma PCSK9 is weakly associated to blood pressure (BP) in large cohorts. Recent evidences suggest that PCSK9 degrades the VLDLR in the adipose tissue. This receptor has been involved in hypoxia-induced lipotoxicity following myocardial infarction.

Objectives:
We aimed at verifying whether PCSK9 affects heart VLDLR content, myocardial function and BP.

Methods:
We used PCSK9
-/-
and PCSK9+/+ mouse littermates (n = 4-6 per group, 3 to 4 month-old males). Two-dimensional LV long-axis and M-mode images were obtained by echocardiography. Mice were also chronically treated with L-NAME (2 mg/kg/d), angiotensin II (minipump, 1 mg/kg/d ) or DOCA-Salt following unilateral nephrectomy.". BP was measured under a 12:12-hour light/dark schedule using a radiotelemetry system or by computerized tail cuff plethysmography.

Results:
Hearts from random-fed PCSK9
-/-
mice didn’t have more VLDLR content (protein and mRNA) than those of PCSK9+/+ mice and had equal TG contents. Fasting (12h) increased myocardial TG contents to the same extent in both genotypes (+ 100%, fed PCSK9++, PCSK9
-/-
vs fasted PCSK9++, PCSK9-/-: 18±5,4 microg/mg, 18,5± 11 microg/mg vs 36±3,5 microg/mg, 38± 5,1 microg/mg, n=3). Despite a ∼6-fold increase in plasma PCSK9 concentrations compared to C57Bl6J mice, hearts from LDLR knockout mice had equal quantities of VLDLR protein. Injecting (i.v.) 125 μg of hPCSK9 in PCSK9+/+ mice (>300 times the physiological concentration) led to an 83% (p<0.05) decrease in mature VLDLR, in the heart and to a >90% decrease of hepatic LDLR, as measured by western blot 1h after the injection. Thus, supra-physiological concentrations of PCSK9 can acutely degrade the VLDLR in mouse heart. The dimensions of the ventricular cavity and the thickness of the septal and the posterior walls as well as LV systolic and diastolic functions were unchanged in PCSK9
-/-
mice. In addition, PCSK9-deficiency did not alter either basal BP or the elevated BP in several models of hypertension.

Collectively these results show that PCSK9 deficiency is neutral on blood pressure or myocardium function at a young age in mice. Plasma PCSK9 at physiological or pathophysiological concentrations do not decrease VLDLR content in the heart.
Abstract 324: Resequencing of the Lipoprotein Lipase Gene in Individuals with Extreme HDL-Cholesterol Levels
Authors: Pirim Dilek , Demirci F Y , Wang Xingbin , Irfan M , Wang Yuee , Hokanson John E , Hamman Richard F , Kammerer Candace M , Kamboh M I
Published: July 2021

In the United States, coronary heart disease (CHD) is the most common cause of death and number one killer of American males and females. Several risk factors for CHD have been identified, including low high-density lipoprotein cholesterol (HDL-C), elevated total cholesterol and low-density lipoprotein cholesterol (LDL-C), and high triglycerides (TG), but the genetic variations underlying inter-individual differences in plasma lipid profiles still need to be further explored. Thus, our interest has been focused on the lipoprotein lipase (
LPL
) which is one of the major genes involved in lipid metabolism. Previously, its sequence variation has been associated with the risk of CHD and other complex traits such as dyslipidemia, type 2 diabetes, essential hypertension, and Alzheimer’s disease. In order to further investigate the common and rare variation in
LPL
, we resequenced the 95 individuals having lower (n=48) and upper (n=48) 5
th
percentiles of HDL-C levels selected from a well defined population-based non-Hispanic White (NHW) sample of 623 individuals. As a result of resequencing the entire
LPL
gene and additional 1kb at the 5’ and 3’ end, a total of 179 variants (substitutions or indels) plus one microsatellite were identified, including 91 relatively uncommon or rare variants [minor allele frequency (MAF) <0.05)] and 88 common variants (MAF ≥0.05). We have observed differences in the distribution of the 91 relatively uncommon or rare variants in two groups; 21 were present only in the low HDL-C group and 25 were present only in the high HDL-C group. The prevalence of uncommon or rare variants was found to be higher in the high HDL-C group. Thirty two common variants showed significantly different (p-value <0.05) allele frequency between the high and low HDL-C groups. To date, we have analyzed 39 common variants (MAF ≥0.05) in the entire sample and our results confirm the previous association findings as well as reveal novel variants that are associated with inter-individual variation in HDL and TG levels. The genotyping effort of relevant rare variants in the entire sample has also been completed and their analysis will help to unravel the role of rare variants in the
LPL
gene in regulation of plasma HDL-C levels and other lipids.
Abstract 325: HDL Binding Rates for MPO: An Index of Functionality?
Authors: Kong Linghong , Sahoo Daisy , Sorci-Thomas Mary , Zhang Hao , Pritchard Kirkwood A
Published: July 2021

Statement of Problem:
Measurements of high density lipoprotein (HDL) function are reported to be more predictive for atheroprotection than measurements of HDL cholesterol. Unfortunately, bioassays of HDL function are complex, manual and time-consuming, making them impractical for routine clinical use. As such, well-standardized and easy-to-use assays of HDL function are desperately needed to make better informed clinical decisions. We are developing a panel of biolayer interferometry (
BLI
) assays on the Octet Red (forteBio, Inc) that measure HDL binding rates for biomolecules that regulate HDL function. Previously we showed that hypochlorous acid-modified HDL (ox-HDL) bound paraoxonase 1, myeloperoxidase (MPO) and cholesteryl ester transfer protein at faster rates than native HDL (n-HDL). Based on these data, we hypothesize that
BLI
assays of HDL binding rates might be useful for assessing functionality
in vivo.

Methods:
To test this hypothesis, the biophysics of HDL interactions with MPO was examined in rodent models of metabolic syndrome and hypercholesterolemia and in patients with documented cardiovascular disease (CVD). Briefly, HDL was immunocaptured onto anti-apoA-I biosensors, incubated in a standardized solution of MPO and binding rates determined in ∼3.5 min/sample.

Results:
HDL from fructose-fed hamsters tended to bind MPO at faster rates than from chow-fed hamsters (300% of control p<0.07, n=6). HDL from hypercholesterolemic

Ldlr
-/-

mice bound MPO at much faster rates than HDL from control mice (175% of control p<0.05, n=4). HDL from patients with CVD bound MPO at faster rates than HDL from control humans (280% of control p<0.004, n=6).

Conclusions:
HDL in hamsters with metabolic syndrome, mice with hypercholesterolemia and patients with CVD appears to bind MPO at faster rates than their corresponding controls. Automated
BLI
assays make it possible to complete large scale clinical studies in a reasonable time frame. Finally biophysical interactions of HDL with biomolecules such as MPO may be a useful approach for quantifying HDL functionality.